Structural and physiological diversity among cystlike resting cells of bacteria of the genus <Emphasis Type="Italic">Pseudomonas</Emphasis>

Cystlike resting cells (CRC) of non-spore-forming gram-negative bacteria of the genus Pseudomonas, P. aurantiaca and P. fluorescens, were obtained and characterized for the first time; their physiological and morphological diversity was demonstrated. The following properties were common for all the revealed types of CRC as dormant forms: (1) long-term (up to 6 months or longer) maintenance of viability in the absence of culture growth and cell respiration; (2) absence of an experimentally detectable level of metabolism; (3) higher resistance to damage and autolysis under the action of provoking factors than in metabolically active vegetative cells; and (4) specific features of ultrastructural organization absent in vegetative cells: thickened and lamellar envelopes, clumpy structure of the cytoplasm, and condensed DNA in nucleoid. The differences in various types of CRC concern the thickness and lamellar structure of cell envelopes, as well as the presence and thickness of the capsular layer. In particular, forms ultrastructurally similar to typical bacterial cysts were revealed in pseudomonad populations growing on soil agar. Physiological diversity was revealed in different levels of viability preservation and thermal resistance in various types of CRC and depended on the conditions of their formation. The optimal conditions and procedures for obtaining P. aurantiaca and P. fluorescens CRC that retain the ability to form colonies on standard nutrient media are as follows: (1) a twofold decrease of nitrogen content in the growth medium; (2) an increased level of anabiosis autoinducer (C₁₂-AHB, 10^?4 M) in stationary cultures; (3) transfer of the cells from stationary cultures to a starvation medium with silica; (4) cultivation in soil extract; and (5) development of cultures on soil agar. The CRC from the cultures grown in soil extract or starvation medium with silica proved to be resistant to heat treatment (60°C, 5 min). In the CRC formed in nitrogen-limited media, the degree of heat resistance increased at longer incubation (1.5 to 6 months). CRCs on soil agar surface were resistant to desiccation. The ultrastructure of the morphologically varied types of P. aurantiaca CRC formed under simulated natural conditions is described for the first time. The data on the intraspecies diversity of pseudomonad dormant forms contribute to the concept of plasticity of the life style and adaptive reactions that ensure survival of these bacteria in unfavorable environmental conditions.     

Monitoring physiological status of GFP-tagged Pseudomonas fluorescens SBW25 under different nutrient conditions and in soil by flow cytometry

Pseudomonas fluorescens SBW25, a plant growth promoting bacterium, has been widely studied due to its potential as an inoculum for improving crop yields. Environmental inoculants are usually applied on seeds or directly to soil and to effectively promote plant growth they need to be viable and active. However, it is difficult to study the physiological status of specific microorganisms in complex environments, such as soil. In this study, our aim was to use molecular tools to specifically monitor the physiological status of P. fluorescens SBW25 in soil and in pure cultures incubated under different nutritional conditions. The cells were previously tagged with marker genes (encoding green fluorescent protein and bacterial luciferase) to specifically track the cells in environmental samples. The physiological status of the cells was determined using the viability stains 5-cyano-2,3-ditolyl-tetrazolium chloride (CTC) and propidium iodide (PI), which stain active and dead cells, respectively. Luciferase activity was used to monitor the metabolic activity of the population. Most of the cells died after incubation for nine days in nutrient rich medium. By contrast when incubated under starvation conditions, most of the population was not stained with CTC or PI (i.e. intact but inactive cells), indicating that most of the cells were presumably dormant. In soil, a large fraction of the SBW25 cell population became inactive and died, as determined by a decline in luciferase activity and CTC-stained cells, an increase in PI-stained cells, and an inability of the cells to be cultured on agar medium. However, approximately 60% of the population was unstained, presumably indicating that the cells entered a state of dormancy in soil similar to that observed under starvation conditions in pure cultures. These results demonstrate the applicability of this approach for monitoring the physiological status of specific cells under stress conditions, such as those experienced by environmental inoculants in soil.     

Diverse morphological types of dormant cells and conditions for their formation in <Emphasis Type="Italic">Azospirillum brasilense</Emphasis>

Differences in generation of dormant forms (DF) were revealed between two strains of non-sporeforming gram-negative bacteria Azospirillum brasilense, Sp7 (non-endophytic) and Sp245 (endophytic strain). In post-stationary ageing bacterial cultures grown in a synthetic medium with a fivefold decreased initial nitrogen content, strain Sp7 formed two types of cyst-like resting cells (CRC). Strain Sp245 did not form such types of DF under the same conditions. CRC of the first type were formed in strain Sp245 only under phosphorus deficiency (C > P). The endophytic strain was also shown to form structurally differentiated cells under complete starvation, i.e. at a transfer of early stationary cultures, grown in the media with C > N unbalance, to saline solution (pH 7.2). These DF had a complex structure similar to that of azotobacter cysts. The CRC, which are generated by both azospirilla strains and belong to distinct morphological types, possessed the following major features: absence of division; specific ultrastructural organization; long-term maintenance of viability (for 4 months and more); higher heat resistance (50–60°C, 10 min) as compared with vegetative cells, i.e. the important criteria for dormant prokaryotic forms. However, CRC of non-endophytic strain Sp7 had higher heat resistance (50, 55, 60°C). The viability maintenance and the portion of heat-resistant cells depended on the conditions of maturation and storage of CRC populations. Long-term storage (for 4 months and more) of azospirilla DF populations at ?20°C was optimal for maintenance of their colony-forming ability (57% of the CFU number in stationary cultures), whereas the largest percentage of heat-resistant cells was in CRC suspensions incubated in a spent culture medium (but not in saline solution) at room temperature. The data on the intraspecies diversity of azospirilla DF demonstrate the relation between certain type DF formation to the type of interaction (non-endophytic or endophytic) with the plant partner and provide more insight into the adaptation mechanisms that ensure the survival of gram-negative non-spore-forming bacteria in nature.     

Effect of cell density and attachment on resuscitation in soil of starved Pseudomonas fluorescens MON787

The effect of cell density and attachment on starvation survival and recovery was determined using luminometry to measure activity of a lux -marked strain of Pseudomonas fluorescens MON787. Bioluminescence was found to be a sensitive indicator of in situ activity of P. fluorescens MON787 in soil. The activity of a bacterial inoculum could be monitored during growth in soil, and was found to correlate with an increase in cell numbers. Luminescence could detect decreasing activity of P. fluorescens during starvation in soil, and recovery of activity and cell numbers following exposure to starvation and matric potential stress. The effect of localised cell density and attachment in soil on recovery from lag phase after nutrient addition was investigated and compared to recovery of starved liquid cultures. Nutrient addition to starved P. fluorescens in soil or liquid medium resulted in an immediate recovery of activity, followed by a second increase in luminescence after 5 h. Cells exposed to both starvation and matric potential stress in soil did not show a detectable immediate increase of activity, but required a 5-h lag phase before recovery of both activity and cell growth. The lag phase values were not significantly different over a range of localised cell densities. This suggests that cell density of P. fluorescens in the range tested is not a factor which affects recovery of soil bacteria from starvation.     

Survival of, and induced stress resistance in, carbon-starved Pseudomonas fluorescens cells residing in soil.

We investigated the survival, cell length, and development of general stress resistance in populations of Pseudomonas fluorescens R2f and its rifampin-resistant mutant, R2f Rpr, following exposure to carbon starvation conditions in liquid cultures and residence in two different soils, Flevo silt loam (FSL) and Ede loamy sand (ELS). In much the same way as was recently shown for P. putida KT2442, carbon-starved P. fluorescens R2f populations revealed enhanced resistance to otherwise lethal treatments, such as exposure to ethanol, high temperature, osmotic tension, and oxidative stress. A large population of nonculturable P. fluorescens R2f Rpr cells arose shortly after their introduction into ELS soil, whereas the formation of nonculturable cells was not observed in FSL soil. Also, the inoculant cell (based on immunofluorescence) and CFU counts decreased faster in ELS soil than in FSL soil. Introduction of carbon-starved instead of exponential-growth-phase R2f Rpr cells into ELS soil did not affect bacterial survival. The inoculant cell length decreased in soil, and no large differences in cell length in the two soil types were observed. Addition of glucose to ELS soil resulted in a stable cell length of R2f Rpr cells, whereas carbon-starved cells introduced into ELS soil remained small. Exponentially growing R2f Rpr cells developed enhanced resistance to ethanol, high temperature, osmotic tension, and oxidative stress within 1 day in both soils, whereas cells introduced into ELS soil amended with glucose showed decreased resistance. Cells that were carbon starved prior to introduction into ELS soil showed unchanged stress resistance levels upon residence in soil.     

Genetic diversity in pseudomonads associated with cereal cultures infected with basal bacteriosis

The genetic properties of 45 pseudomonad strains isolated from cereal cultures exhibiting symptoms of basal bacteriosis have been investigated. Considerable genetic diversity has been demonstrated using DNA fingerprints obtained by amplification with REP, ERIC, and BOX primers. Restriction analysis of the 16S-23S internal transcribed spacer (ITS1) allowed the strains to be subdivided into two major groups. In a phylogenetic tree, the ITS1s of these groups fell into two clusters, which also included the ITS1 of Pseudomonas syringae ("Syringae" cluster) and the ITS1 of P. fluorescens, P. tolaasii, P. reactans, P. gingeri, and P. agarici ("Fluorescens" cluster) from the GenBank database. Comparison of the ITS1 divergence levels within the "Fluorescens" cluster suggests expediency of treating P. tolaasii, P. reactans, various P. fluorescens groups, and, possibly, P. gingeri and P. agarici as subspecies of one genospecies. The intragenomic heterogeneity of ITS1s was observed in some of the pseudomonad strains studied. The results of amplification with specific primers and subsequent sequencing of the amplificate suggest the possibility of the presence of a functionally active syrB gene involved in syringomycin biosynthesis in the strains studied.     

Effect of alkylhydroxybenzenes, microbial anabiosis inducers, on the structural organization of Pseudomonas aurantiaca DNA and on phenotypic dissociation induction

We revealed a relationship between alkylhydroxybenzene (AHB)-induced changes in the structural organization of supramolecular complexes (SC) of the DNA of Pseudomonas auraniaca and the phenotypic dissociation of this bacterium. The addition of 0.1-0.3 mM hexylresorcinol (C6-AHB), a chemical analogue of microbial anabiosis autoinducers, caused the formation of cystlike refractile cells (CRC) in these gram-negative, nonsporulating bacteria. Inoculating pseudomonad CRC on solid nutrient media resulted in phenotypic dissociation of the microbial population that yielded several variants with different colony structure and morphology. This manifested itself in the conversion of the original S-colony-forming phenotype into the R form and in the formation of less pigmented colonies. These transitions were possibly linked to AHB-induced structural changes in the DNA. In vitro studies revealed that AHB could interact with DNA SC, resulting in their structural modification that manifested itself in changes in their elastoviscosity. DNA supramolecular complexes isolated from proliferating, stationary-phase, and anabiotic P. aurantiaca cells differed in their elastoviscosity and capacity to interact with AHB homologues with different hydrophobicity, such as hexylresorcinol and methylresorcinol (C1-AHB). The DNA SC from actively proliferating cells were characterized by smaller elastoviscosity compared with those from stationary-phase and anabiotic cells, due to the difference in the DNA superspiralization degree and the physiological age of the bacteria involved. C6-AHB produced a pronounced relaxing effect on the DNA SC from exponential-phase P. aurantiaca cells. The less hydrophobic C1-AHB produced a similar effect on the DNA SC from stationary-phase cells. The curve of the dose-effect dependence of C6-AHB had a breaking point within the submillimolar (10(-4) M) concentration range. These concentrations induce the formation of cystlike anabiotic pseudomonad cells that are characterized by an unstable genotype and dissociate into distinct variants upon inoculation on solid media.     

Pseudomonas fluorescens DR54 Reduces Sclerotia Formation,Biomass Development,and Disease Incidence of Rhizoctonia solani Causing Damping-Off in Sugar Beet

Effects of the biocontrol strain, Pseudomonas fluorescens DR54, on growth and disease development by Rhizoctonia solani causing damping-off in sugar beet were studied in soil microcosms and in pot experiments with natural, clay-type soil. In pot experiments with P. fluorescens DR54-treated seeds, significantly fewer Rhizoctonia-challenged seedlings showed damping-off symptoms than when not inoculated with the biocontrol agent. In the rhizosphere of P. fluorescens DR54 inoculated seeds, the bacterial inoculant was present in high numbers as shown by dilution plating and immunoblotting. By the ELISA antibody technique and direct microscopy of the fungal pathogen grown in soil microcosms, it was shown that the presence of P. fluorescens DR54 on the inoculated seeds had a strong inhibitory effect on development of both mycelium biomass and sclerotia formation by R. solani. In the field experiment, plant emergence was increased by treatment with P. fluorescens DR54 and the inoculant was found to be the dominating rhizosphere colonizing pseudomonad immediately after seedling emergence.     

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.     

Pseudomonas fluorescens enhances resistance and natural enemy population in rice plants against leaffolder pest

Plant growth promoting fluorescent pseudomonad strains Pf1, TDK1 and PY15 were evaluated for their efficacy against leaffolder ( Cnaphalocrocis medinalis ) pest in rice plants under field conditions individually and in combinations. Application of mixtures of Pseudomonas fluorescens strains Pf1, TDK1 and PY15 significantly reduced the leaffolder damage in rice plants compared with untreated control. Interestingly, natural enemy population in plots treated with P. fluorescens was greater than the chemical and untreated controls. Further, support for these results was gathered by assaying activities of polyphenol oxidase (PPO) and lipoxygenase (LOX) under glasshouse conditions. The results showed the higher activity of PPO and LOX in plants treated with P. fluorescens mixtures (Pf1 + TDK1 + PY15) than the plants treated with individual strains, chemical and untreated controls. Further, fluorescent pseudomonad mixtures increased the rice yield compared with individual strain and non-bacterized treatments. The present study reveals that in addition to plant growth promotion, plant growth-promoting rhizobacterial (PGPR) strains-mediated induction of PPO and LOX in rice plants could be involved in enhanced natural enemy populations and resistance mechanisms against leaffolder attack.     

Pseudomonas fluorescens subsp. cellulosa: an alternative model for bacterial cellulase

G.P. HAZLEWOOD, J.I. LAURIE, L.M.A. FERREIRA AND H.J. GILBERT. 1992. Pseudomonas fluorescens subsp. cellulosa , a Gram-negative soil bacterium, can utilize crystalline cellulose or xylan as main sources of carbon and energy. Synthesis of endoglucanases and xylanases is induced by Avicel, filter paper, carboxymethylcellulose or xylan and is repressed by cellobiose, glucose or xylose. These enzymes are secreted into the culture supernatant fluid and do not form aggregates or associate with the cell surface. Cells of Ps. fluorescens subsp. cellulosa do not adhere to cellulose. In cultures containing Avicel or filter paper, a significant proportion of the secreted cellulase and xylanase activities becomes tightly bound to the insoluble cellulose. Western blotting has revealed that endoglucanase B, xylanase A and a cellodextrinase encoded by genes previously isolated from Ps. fluorescens subsp. cellulosa and expressed in Escherichia coli , are synthesized by the pseudomonad under a variety of conditions. These enzymes appear to be post-translationally modified, probably through glycosylation. Overall, it appears that the cellulase/hemicellulase system of Ps. fluorescens subsp. cellulosa differs from the model established for celluloytic anaerobes such as Clostridium thermocellum.     

Pseudomonas fluorescens subsp. cellulosa: an alternative model for bacterial cellulase.

Pseudomonas fluorescens subsp. cellulosa, a Gram-negative soil bacterium, can utilize crystalline cellulose or xylan as main sources of carbon and energy. Synthesis of endoglucanases and xylanases is induced by Avicel, filter paper, carboxymethylcellulose or xylan and is repressed by cellobiose, glucose or xylose. These enzymes are secreted into the culture supernatant fluid and do not form aggregates or associate with the cell surface. Cells of Ps. fluorescens subsp. cellulosa do not adhere to cellulose. In cultures containing Avicel or filter paper, a significant proportion of the secreted cellulase and xylanase activities becomes tightly bound to the insoluble cellulose. Western blotting has revealed that endoglucanase B, xylanase A and a cellodextrinase encoded by genes previously isolated from Ps. fluorescens subsp. cellulosa and expressed in Escherichia coli, are synthesized by the pseudomonad under a variety of conditions. These enzymes appear to be post-translationally modified, probably through glycosylation. Overall, it appears that the cellulase/hemicellulase system of Ps. fluorescens subsp. cellulosa differs from the model established for celluloytic anaerobes such as Clostridium thermocellum.     

Inhibition and Agglutination of Arthrobacters by Pseudomonads

The bacterial flora of water in Narragansett Bay, R.I., was observed semimonthly from 1962 to 1964. Dominant isolates were keyed to genus, and the isolates for each genus were expressed as percentage of total isolates. There was a consistent inverse relationship between arthrobacters and the dominant pseudomonads. Pseudomonad growth on agar plates markedly inhibited arthrobacter cross-streaks. Agar from inhibition zones as well as supernatant fluids from pseudomonad broth cultures inhibited arthrobacter motility and caused the cells to agglutinate. Gummy pseudomonad residues from vacuum-evaporated broth cultures readily passed a G-25 Sephadex column. This material agglutinated arthrobacter cells, but failed to cause arthrobacter inhibition in filter-pad assays. In contrast, sterile medium inside a dialysis sac, inoculated externally with a pseudomonad, was inhibitory to arthrobacters in pad assay but failed to agglutinate arthrobacter cells. Pseudomonad isolates from soil showed similar inhibiting and agglutinating activities for both soil and seawater arthrobacter isolates. The inhibitory and agglutinating activities of pseudomonad isolates appeared to diminish on prolonged laboratory cultivation.     

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.     

Soil bacterial DNA and biovolume profiles measured by flow-cytometry

Abstract Flow-cytometry was used to measure cell volumes and DNA contents of single cells in cultures of soil bacteria during exponential growth and starvation conditions. DNA was measured after staining with mitramycin/ethidium bromide. The measurement of DNA was calibrated with rifampicin-treated cells of E. coli containing even numbers of genomes per cell. Cell volumes were assessed by scatter light measurements. Constant DNA to biovolume relations over a range of cell sizes were found for each of the bacteria at exponential growth, and DNA contents per cell varied over a range equivalent to 1–4 genomes per cell. At generation times of 1.0–1.5 h, two genomes were registered as a mean. After starvation of washed cells in a salt solution (24 hrs), a fraction of the cells in each culture had DNA contents equivalent to 1 genome, but significant fractions retained DNA contents equivalent to 2–4 genomes. Attempts to create cells with even numbers of genomes per cell by treatment with rifampicin was successful on an Acinetobacter sp. In contrast, the response to rifampicin was less clear for Pseudomonas fluorescens and P. chlororaphis , and unclear for the gram positive bacteria isolated from soil. The mean decrease in biovolume upon starvation was 4.1 times (range 1.3–8.1 times) and larger than the mean decrease in DNA content of 1.8 (range 1.3–2.7 times). Cell volume determinations by measurements of scatter light was compared with volume determinations by fluorescence microscopy. The amounts of scatter light per volumes was variable, not only did we find large differences between bacterial types, but also between starving and exponentially growing cells of the same isolate. In order to use light scatter as a measure of biovolume, internal standards has to be chosen of comparable size and surface properties as to soil bacteria.     

Intraspecies diversity of dormant forms of <Emphasis Type="Italic">Mycobacterium smegmatis</Emphasis>

The non-spore-forming gram-positive bacterium Mycobacterium smegmatis mc² 155, related to M. tuberculosis, was revealed to be capable of forming different types of dormant forms (DFs) during the life cycle of its cultures. The relationship between the intraspecies diversity of DFs and the cultivation conditions of the mycobacterium was established. The DFs possessed the following common properties: (i) maintenance of viability for a long period of time (5 months), (ii) resistance to deleterious factors such as heat treatment, and (iii) morphological and ultrastructural peculiarities that distinguish DFs from vegetative cells. The diversity of M. smegmatis DFs manifested itself in differences in terms of structural organization, conditions required for growth renewal, and capacity to produce antibiotic-resistant variants upon germination on selective media. Well-differentiated cystlike dormant cells (CDCs) were formed in the cultures grown in synthetic SR1 medium with fivefold-decreased nitrogen content. The structural organization of CDCs differed from that of other DF types mainly in the presence of club-shaped cells, thickened lamellar cell walls, coarse cytoplasm texture, and large electron-transparent triacylglyceride inclusion bodies. It was possible to use mycobacterial CDCs as a source of PCR-competent DNA. CDC populations were heterogeneous in cell buoyant density, and the individual fractions, which we isolated, were found to differ in thermal stability and the ability to revert to growth under standard conditions. Coccoid DFs, which retained their colony-forming capacity for a long time but were less heat-resistant than the CDCs, were formed by mycobacteria grown in standard Sauton’s medium with initial pH value decreased to 6.2. Poorly differentiated DFs resulted from growing mycobacterial cultures in Sauton’s medium with a fivefold-decreased phosphorus content. Upon germination of various DF types, the variants resistant to kanamycin (200 μg/ml) and tetracycline (20 μg/ml) were obtained. CDC suspensions incubated for 5 months demonstrated the highest percentage (1.5%) of antibiotic-resistant clones. The data obtained on the DF diversity of M. smegmatis, a fast-growing relative of M. tuberculosis, contribute to our understanding of the flexibility of the survival strategy of this bacterium in nature and in the host organism.     

Survival and Phospholipid Fatty Acid Profiles of Surface and Subsurface Bacteria in Natural Sediment Microcosms

Although starvation survival has been characterized for many bacteria, few subsurface bacteria have been tested, and few if any have been tested in natural subsurface porous media. We hypothesized that subsurface bacteria may be uniquely adapted for long-term survival in situ. We further hypothesized that subsurface conditions (sediment type and moisture content) would influence microbial survival. We compared starvation survival capabilities of surface and subsurface strains of Pseudomonas fluorescens and a novel Arthrobacter sp. in microcosms composed of natural sediments. Bacteria were incubated for up to 64 weeks under saturated and unsaturated conditions in sterilized microcosms containing either a silty sand paleosol (buried soil) or a sandy silt nonpaleosol sediment. Direct counts, plate counts, and cell sizes were measured. Membrane phospholipid fatty acid (PLFA) profiles were quantified to determine temporal patterns of PLFA stress signatures and differences in PLFAs among strains and treatments. The Arthrobacter strains survived better than the P. fluorescens strains; however, differences in survival between surface and subsurface strains of each genus were not significant. Bacteria survived better in the paleosol than in the nonpaleosol and survived better under saturated conditions than under unsaturated conditions. Cell volumes of all strains decreased; however, sediment type and moisture did not influence rates of miniaturization. Both P. fluorescens strains showed PLFA stress signatures typical for gram-negative bacteria: increased ratios of saturated to unsaturated fatty acids, increased ratios of trans- to cis-monoenoic fatty acids, and increased ratios of cyclopropyl to monoenoic precursor fatty acids. The Arthrobacter strains showed few changes in PLFAs. Environmental conditions strongly influenced PLFA profiles.     

Membrane Filter Method for the Isolation and Enumeration of Pseudomonas aeruginosa from Swimming Pools

A membrane filter technique using black membrane filters, MacConkey agar and fluorescence under ultraviolet (UV) light was investigated for the quantitative isolation of Pseudomonas aeruginosa from swimming pools. Three thousand four hundred forty-five samples were collected from public swimming pools and enumerated by this method over a 6-month period. Fluorescent cultures were isolated from 222 specimens. Seventy-seven of these fluorescent cultures were selected for biochemical screening, with 75 (97%) being verified as P. aeruginosa. To further assess the specificity and sensitivity of this UV screening technique, a comparative study was made of some morphological and biochemical characteristics of fluorescent pseudomonads obtained from different sources. The sensitivity of the method was unimpaired by either colony types or biochemical variations of P. aeruginosa. The failure of the other two fluorescent species, P. fluorescens and P. putida, to grow and/or fluoresce on MacConkey agar at 37 C illustrates the specificity of this technique. Further studies are needed to compare the viability of P. aeruginosa on MacConkey agar to that of efficacious nonselective media.     

Extracellular factors of adaptation of Pneudomonas fluorescens batch cultures to adverse conditions

The culture liquid filtrate of an exponential-phase Pseudomonas fluorescens batch culture added to another P. fluorescens culture at the moment of inoculation was found (1) to prevent or diminish cell adsorption of the flask walls; (2) to enhance the intensity of cell respiration; (3) to shorten the period of adaptation of LB-grown cells to growth in glucose-containing mineral M9 medium; (4) to stimulate bacterial growth at supraoptimum temperature (36 degrees C) and pH values (4.8 and 9.2); and (5) to decrease the death rate of bacteria at the supraoptimum growth temperature. These results were interpreted as indicating that P. fluorescens cultures produce two types of regulatory exometabolites similar to those revealed earlier in Escherichia coli and Bacillus subtilis cultures: the direct-action adaptogenic factor XI capable of increasing bacterial resistance to unfavorable growth conditions (temperature and pH) and factor promoting adaptation to new media. Both factors are presumably low-molecular-weight hydrophilic nonprotein compounds.