Bacterial Dispersers along Preferential Flow Paths of a Clay Till Depth Profile.

This study assessed the dispersal of five bacterial communities from contrasting compartments along a fractured clay till depth profile comprising plow layer soil, preferential flow paths (biopores and the tectonic fractures below), and matrix sediments, down to 350 cm below the surface. A recently developed expansion of the porous surface model (PSM) was used to capture bacterial communities dispersing under controlled hydration conditions on a soil-like surface. All five communities contained bacteria capable of active dispersal under relatively low hydration conditions (-3.1 kPa). Further testing of the plow layer community revealed active dispersal even at matric potentials of -6.3 to -8.4 kPa, previously thought to be too dry for dispersal on the PSM. Using 16S rRNA gene amplicon sequencing, the dispersing communities were found to be less diverse than their corresponding total communities. The dominant dispersers in most compartments belonged to the genus Pseudomonas and, in the plow layer soil, to Rahnella as well. An exception to this was the dispersing community in the matrix at 350 cm below the surface, which was dominated by Pantoea Hydrologically connected compartments shared proportionally more dispersing than nondispersing amplicon sequence variants (ASVs), suggesting that active dispersal is important for colonizing these compartments. These results highlight the importance of including soil profile heterogeneity when assessing the role of active dispersal and contribute to discerning the importance of active dispersal in the soil environment.IMPORTANCE The ability to disperse is considered essential for soil bacteria colonization and survival, yet very little is known about the dispersal ability of communities from different heterogeneous soil compartments. Important factors for dispersal are the thickness and connectivity of the liquid film between soil particles. The present results from a fractured clay till depth profile suggest that dispersal ability is common in various soil compartments and that most are dominated by a few dispersing taxa. Importantly, an increase in shared dispersers among the preferential flow paths of the clay till suggests that active dispersal plays a role in the successful colonization of these habitats.

Novel Method Reveals a Narrow Phylogenetic Distribution of Bacterial Dispersers in Environmental Communities Exposed to Low-Hydration Conditions.

In this study, we developed a method that provides profiles of community-level surface dispersal from environmental samples under controlled hydration conditions and enables us to isolate and uncover the diversity of the fastest bacterial dispersers. The method expands on the porous surface model (PSM), previously used to monitor the dispersal of individual bacterial strains in liquid films at the surface of a porous ceramic disc. The novel procedure targets complex communities and captures the dispersed bacteria on a solid medium for growth and detection. The method was first validated by distinguishing motile Pseudomonas putida and Flavobacterium johnsoniae strains from their nonmotile mutants. Applying the method to soil and lake water bacterial communities showed that community-scale dispersal declined as conditions became drier. However, for both communities, dispersal was detected even under low-hydration conditions (matric potential, -3.1 kPa) previously proven too dry for P. putida strain KT2440 motility. We were then able to specifically recover and characterize the fastest dispersers from the inoculated communities. For both soil and lake samples, 16S rRNA gene amplicon sequencing revealed that the fastest dispersers were substantially less diverse than the total communities. The dispersing fraction of the soil microbial community was dominated by Pseudomonas species cells, which increased in abundance under low-hydration conditions, while the dispersing fraction of the lake community was dominated by Aeromonas species cells and, under wet conditions (-0.5 kPa), also by Exiguobacterium species cells. The results gained in this study bring us a step closer to assessing the dispersal ability within complex communities under environmentally relevant conditions.IMPORTANCE Dispersal is a key process of bacterial community assembly, and yet, very few attempts have been made to assess bacterial dispersal at the community level, as the focus has previously been on pure-culture studies. A crucial factor for dispersal in habitats where hydration conditions vary, such as soils, is the thickness of the liquid films surrounding solid surfaces, but little is known about how the ability to disperse in such films varies within bacterial communities. Therefore, we developed a method to profile community dispersal and identify fast dispersers on a rough surface resembling soil surfaces. Our results suggest that within the motile fraction of a bacterial community, only a minority of the bacterial types are able to disperse in the thinnest liquid films. During dry periods, these efficient dispersers can gain a significant fitness advantage through their ability to colonize new habitats ahead of the rest of the community.

Copper amendment of agricultural soil selects for bacterial antibiotic resistance in the field.

AIMS: The objective of this study was to determine whether Cu-amendment of field plots affects the frequency of Cu resistance, and antibiotic resistance patterns in indigenous soil bacteria. METHODS AND RESULTS: Soil bacteria were isolated from untreated and Cu-amended field plots. Cu-amendment significantly increased the frequency of Cu-resistant isolates. A panel of isolates were characterized by Gram-reaction, amplified ribosomal DNA restriction analysis and resistance profiling against seven antibiotics. More than 95% of the Cu-resistant isolates were Gram-negative. Cu-resistant Gram-negative isolates had significantly higher incidence of resistance to ampicillin, sulphanilamide and multiple (> or =3) antibiotics than Cu-sensitive Gram-negative isolates. Furthermore, Cu-resistant Gram-negative isolates from Cu-contaminated plots had significantly higher incidence of resistance to chloramphenicol and multiple (> or =2) antibiotics than corresponding isolates from control plots. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this field experiment show that introduction of Cu to agricultural soil selects for Cu resistance, but also indirectly selects for antibiotic resistance in the Cu-resistant bacteria. Hence, the widespread accumulation of Cu in agricultural soils worldwide could have a significant effect on the environmental selection of antibiotic resistance.

Input of protein to lake water microcosms affects expression of proteolytic enzymes and the dynamics of Pseudomonas spp.

The objective of this study was to determine how an input of protein to lake water affects expression of a proteolytic potential and influences the abundance and composition of a specific group of bacteria. Pseudomonas spp. were chosen as a target group that can be recovered on selective growth media and contain both proteolytic and nonproteolytic strains. Amendment with 2 mg of casein per liter increased total proteinase activity (hydrolysis of [(3)H]casein) by 74%, leucine-aminopeptidase activity (hydrolysis of leucine-methyl-coumarinylamide) by 133%, bacterial abundance by 44%, and phytoplankton biomass (chlorophyll a) by 39%. The casein amendment also increased the abundance of culturable Pseudomonas spp. by fivefold relative to control microcosms but did not select for proteolytic isolates. Soluble proteins immunochemically related to the Pseudomonas fluorescens alkaline proteinase, AprX, were detected in amended microcosms but not in the controls. The expression of this class of proteinase was confirmed exclusively for proteolytic Pseudomonas isolates from the microcosms. The population structure of Pseudomonas isolates was determined from genomic fingerprints generated by universally primed PCR, and the analysis indicated that casein amendment led to only minor shifts in population structure. The appearance of AprX-like proteinases in the lake water might thus reflect a general induction of enzyme expression rather than pronounced shifts in the Pseudomonas population structure. The limited effect of casein amendment on Pseudomonas population structure might be due to the availability of casein hydrolysates to bacteria independent of their proteinase expression. In the lake water, 44% of the total proteinase activity was recovered in 0.22-microm-pore-size filtrates and thus without a direct association with the bacteria providing the extracellular enzyme activity. Since all Pseudomonas isolates expressed leucine-aminopeptidase in pure culture, proteolytic as well as nonproteolytic pseudomonads were likely members of the bacterial consortium that metabolized protein in the lake water.

An altered Pseudomonas diversity is recovered from soil by using nutrient-poor Pseudomonas-selective soil extract media.

We designed five Pseudomonas-selective soil extract NAA media containing the selective properties of trimethoprim and sodium lauroyl sarcosine and 0 to 100% of the amount of Casamino Acids used in the classical Pseudomonas-selective Gould's S1 medium. All of the isolates were confirmed to be Pseudomonas by a Pseudomonas-specific OprF antibody and a Pseudomonas-specific PCR targeting 16S ribosomal DNA. The Pseudomonas isolates were characterized by classical physiological tests, repetitive extragenic palindromic-PCR, Fourier transform infrared spectroscopy, and carbon source utilization patterns. Several of these analyses showed that the amount of Casamino Acids significantly influenced the diversity of the recovered Pseudomonas isolates. Furthermore, the data suggested that specific Pseudomonas subpopulations were represented on the nutrient-poor media. The NAA 1:100 medium, containing ca. 15 mg of organic carbon per liter, consistently gave significantly higher Pseudomonas CFU counts than Gould's S1 when tested on four Danish soils. NAA 1:100 may, therefore, be a better medium than Gould's S1 for enumeration and isolation of Pseudomonas from the low-nutrient soil environment.

Carbon limitation induces sigma(S)-dependent gene expression in Pseudomonas fluorescens in soil.

Recent studies employing reporter gene technology indicate that the availabilities of the major nutrients nitrogen, phosphate, and iron to Pseudomonas are not severely limited in bulk soil. Indirect evidence has pointed to carbon limitation as a severe nutritional stress in this environment. We show that a plasmid (pGM115)-borne transcriptional fusion between the sigma(S)-dependent Escherichia coli promoter P(fic) and lacZ functions as a reliable reporter for carbon availability in Pseudomonas fluorescens. When P. fluorescens strain DF57(pGM115) was introduced into bulk soil, carbon-limiting conditions were indicated by citrate-repressible induction of beta-galactosidase activity. To address carbon availability at the single-cell level, we developed an immunofluorescence double-staining procedure for individual DF57 cells expressing beta-galactosidase from P(fic). Changes in cell size and expression of beta-galactosidase were analyzed by flow cytometry. Cells extracted from soil microcosms reduced their size less than carbon-starved cells in pure culture and showed an increased tendency to aggregate. The single-cell analysis revealed that for cells residing in soil, the expression of beta-galactosidase became heterogeneous and only a DF57 subpopulation appeared to be carbon limited. In soil amended with barley straw, limited nitrogen availability has been determined by use of the bioluminescent reporter strain P. fluorescens DF57-N3. We used strain DF57-N3(pGM115) as a double reporter for carbon and nitrogen limitation that allowed us to study the dynamics of carbon and nitrogen availabilities in more detail. In straw-amended soil beta-galactosidase activity remained low, while nitrogen limitation-dependent bioluminescence appeared after a few days. Hence, nitrogen became limited under conditions where carbon resources were not completely exhausted.

A panel of Tn7-based vectors for insertion of the gfp marker gene or for delivery of cloned DNA into Gram-negative bacteria at a neutral chromosomal site.

The use of Tn7-based systems for site-specific insertion of DNA into the chromosome of Gram-negative bacteria has been limited due to the lack of appropriate vectors. We therefore developed a flexible panel of Tn7 delivery vectors. In one group of vectors, the miniTn7 element, which is inserted into the chromosome, contains a multiple cloning site (MCS) and the kanamycin, streptomycin or gentamicin resistance markers. Another group of vectors intended for tagging with green fluorescent protein (GFP) carries the gfpmut3* gene controlled by the modified lac promoter PA1/04/03, several transcriptional terminators, and various resistance markers. These vectors insert Tn7 into a specific, neutral intergenic region immediately downstream of the gene encoding glucosamine-6-phosphate synthetase (GlmS) in the tested fluorescent Pseudomonas strains. The gfp-tagging vector containing a gentamicin-resistance marker is useful for tagging strains carrying a Tn5 transposon. Tn5 transposons often carry kanamycin-resistance-encoding genes and are frequently used to generate bacterial mutants and to deliver reporter constructions in gene expression studies. To demonstrate the utility of a dual marker/reporter system, the Tn7-gfp marker system was combined with a Tn5-delivered luxAB reporter system in Pseudomonas fluorescens. The system allowed detection of gfp-tagged cells in the barley rhizosphere, while expression of the Tn5-tagged locus could be determined by measuring bioluminescence.

Interactions between proteolytic and non-proteolytic Pseudomonas fluorescens affect protein degradation in a model community.

The metabolic interactions between proteinase-producing bacteria and other members of bacterial communities are poorly investigated, although they are important for the understanding of structure-function relationships in complex ecosystems. We constructed simple model communities consisting of proteolytic and non-proteolytic Pseudomonas fluorescens strains to identify relevant interactions and to assess their specific significance during the mobilization of protein for growth. The proteolytic or non-proteolytic model communities were established by co-inoculating proteolytic or proteinase-deficient Tn5-mutants of P. fluorescens strain ON2 with the non-proteolytic reporter strain DF57-N3 that expresses bioluminescence in response to nitrogen limitation. The growth medium was composed such that growth would be nitrogen limited in the absence of proteolytic activity. In the proteolytic communities data on growth and nitrogen availability showed that the protein hydrolysates were available to both the proteolytic and the non-proteolytic strain. Competition between these strains profoundly affected both growth and proteinase production. Hence, the mobilization of protein was closely coupled to the competitive success of the proteolytic strain. These findings provide new insight into the metabolic interactions that occur when protein is degraded in mixed bacterial communities.

Emergency derepression: stringency allows RNA polymerase to override negative control by an active repressor.

The uspA promoter, driving production of the universal stress protein A in response to diverse stresses, is demonstrated to be under dual control. One regulatory pathway involves activation of the promoter by the alarmone guanosine 3',5'-bisphosphate, via the beta-subunit of RNA polymerase, whereas the other consists of negative control by the FadR repressor. In contrast to canonical dual control by activation and repression circuits, which depends on concomitant activation and derepression for induction to occur, the ppGpp-dependent activation of the uspA promoter overrides repression by an active FadR under conditions of severe cellular stress (starvation). The ability of RNA polymerase to overcome repression during stringency depends, in part, on the strength of the FadR operator. This emergency derepression is operative on other FadR-regulated genes induced by starvation and is argued to be an essential regulatory mechanism operating during severe stress.

Nitrogen availability to Pseudomonas fluorescens DF57 is limited during decomposition of barley straw in bulk soil and in the barley rhizosphere.

The availability of nitrogen to Pseudomonas fluorescens DF57 during straw degradation in bulk soil and in barley rhizosphere was studied by introducing a bioluminescent reporter strain (DF57-N3), responding to nitrogen limitation, to model systems of varying complexity. DF57-N3 was apparently not nitrogen limited in the natural and sterilized bulk soil used for these experiments. The soil was subsequently amended with barley straw, representing a plant residue with a high carbon-to-nitrogen ratio (between 60 and 100). In these systems the DF57-N3 population gradually developed a nitrogen limitation response during the first week of straw decomposition, but exclusively in the presence of the indigenous microbial population. This probably reflects the restricted ability of DF57 to degrade plant polymers by hydrolytic enzymes. The impact of the indigenous population on nitrogen availability to DF57-N3 was mimicked by the cellulolytic organism Trichoderma harzianum Rifai strain T3 when coinoculated with DF57-N3 in sterilized, straw-amended soil. Limitation occurred concomitantly with fungal cellulase production, pointing to the significance of hydrolytic activity for the mobilization of straw carbon sources, thereby increasing the nitrogen demand. Enhanced survival of DF57-N3 in natural soil after straw amendment further indicated that DF57 was cross-fed with carbon/energy sources. The natural barley rhizosphere was experienced by DF57-N3 as an environment with restricted nitrogen availability regardless of straw amendment. In the rhizosphere of plants grown in sterilized soil, nitrogen limitation was less severe, pointing to competition with indigenous microorganisms as an important determinant of the nitrogen status for DF57-N3 in this environment. Hence, these studies have demonstrated that nitrogen availability and gene expression in Pseudomonas is intimately linked to the structure and function of the microbial community. Further, it was demonstrated that the activities of cellulolytic microorganisms may affect the availability of energy and specific nutrients to a group of organisms deficient in hydrolytic enzyme activities.

Green fluorescent protein-marked Pseudomonas fluorescens: localization, viability, and activity in the natural barley rhizosphere.

The gfp-tagged Pseudomonas fluorescens biocontrol strain DR54-BN14 was introduced into the barley rhizosphere. Confocal laser scanning microscopy revealed that the rhizoplane populations of DR54-BN14 on 3- to 14-day-old roots were able to form microcolonies closely associated with the indigenous bacteria and that a majority of DR54-BN14 cells appeared small and almost coccoid. Information on the viability of the inoculant was provided by a microcolony assay, while measurements of cell volume, the intensity of green fluorescent protein fluorescence, and the ratio of dividing cells to total cells were used as indicators of cellular activity. At a soil moisture close to the water-holding capacity of the soil, the activity parameters suggested that the majority of DR54-BN14 cells were starving in the rhizosphere. Nevertheless, approximately 80% of the population was either culturable or viable but nonculturable during the 3-week incubation period. No impact of root decay on viability was observed, and differences in viability or activity among DR54-BN14 cells located in different regions of the root were not apparent. In dry soil, however, the nonviable state of DR54-BN14 was predominant, suggesting that desiccation is an important abiotic regulator of cell viability.

Distribution of metabolic activity and phosphate starvation response of lux-tagged Pseudomonas fluorescens reporter bacteria in the barley rhizosphere.

The purpose of this study was to determine the metabolic activity of Pseudomonas fluorescens DF57 in the barley rhizosphere and to assess whether sufficient phosphate was available to the bacterium. Hence, two DF57 reporter strains carrying chromosomal luxAB gene fusions were introduced into the rhizosphere. Strain DF57-40E7 expressed luxAB constitutively, making bioluminescence dependent upon the metabolic activity of the cells under defined assay conditions. The DF57-P2 reporter strain responded to phosphate limitation, and the luxAB gene fusion was controlled by a promoter containing regulatory sequences characteristic of members of the phosphate (Pho) regulon. DF57 generally had higher metabolic activity in a gnotobiotic rhizosphere than in the corresponding bulk soil. Within the rhizosphere the distribution of metabolic activity along the root differed between the rhizosphere soil and the rhizoplane, suggesting that growth conditions may differ between these two habitats. The DF57-P2 reporter strain encountered phosphate limitation in a gnotobiotic rhizosphere but not in a natural rhizosphere. This difference in phosphate availability seemed to be due to the indigenous microbial population, as DF57-P2 did not report phosphate limitation when established in the rhizosphere of plants in sterilized soil amended with indigenous microorganisms.

Growth and viability of Alcaligenes eutrophus JMP134 in seawater as affected by substrate and nutrient amendment.

Growth and viability of Alcaligenes eutrophus JMP134 was studied in laboratory microcosms with 0.2 microns-filtered seawater prior to release in field-based mesocosms. In unamended systems JMP134 did not grow and viability, measured as direct viable counts combined with immunofluorescence microscopy, was 40-50%. Addition of a nitrogen + phosphorus nutrient mixture caused a greater growth response than amendment with a carbon substrate. Amendment with substrate and/or nutrients caused an increase in viability to ca 100% but only for a brief period coinciding with cell proliferation. Hence, Alc. eutrophus JMP134 has a limited survival potential in seawater unless it is supplied with additional nutrients.

Outer Membrane Protein Heterogeneity within Pseudomonas fluorescens and P. putida and Use of an OprF Antibody as a Probe for rRNA Homology Group I Pseudomonads.

The electrophoretic patterns of outer membrane proteins of strains representing the biovars of Pseudomonas fluorescens and Pseudomonas putida were analyzed by gel electrophoresis. The outer membrane protein profiles were variable, and they were not useful for assigning strains to a specific biovar. However, three or four predominant outer membrane proteins migrating at 42 to 46 kDa, 33 to 38 kDa, and 20 to 22 kDa were conserved among the strains. They could be tentatively identified as OprE (44 kDa), OprF (38 kDa), OprH (21 kDa), and OprL (20.5 kDa), which are known proteins from Pseudomonas aeruginosa. A 37-kDa OprF-like protein was purified from P. fluorescens DF57 and used to raise a polyclonal antibody. In Western blot (immunoblot) analysis, this antibody reacted with OprF proteins from members of Pseudomonas rRNA homology group I but not with proteins from nonpseudomonads. The heterogeneity in M(infr) of OprF was greater among P. fluorescens strains than among P. putida strains. Immunofluorescence microscopy of intact cells demonstrated that the antibody recognized epitopes that were accessible only after unmasking by EDTA treatment. The antibody was used in a colony blotting assay to determine the percentage of rRNA homology group I pseudomonads among bacteria from the rhizosphere of barley. The bacteria were isolated on 10% tryptic soy agar, King's B agar, and the pseudomonad-specific medium Gould S1 agar. The estimate of OprF-containing CFU in rhizosphere soil obtained by colony blotting on 10% tryptic soy agar was about 2 and 14 times higher than the values obtained from King's agar and Gould S1 agar, respectively, indicating that not all fluorescent pseudomonads are scored on more specific media. The colonies reacting with the OprF antibody were verified as being rRNA homology group I pseudomonads by using the API 20NE system.

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

Mesocosms (∼4.5 m(3)) 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 × 10(5) 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.

Culturability and Expression of Outer Membrane Proteins during Carbon, Nitrogen, or Phosphorus Starvation of Pseudomonas fluorescens DF57 and Pseudomonas putida DF14.

Changes in culturability and outer membrane protein profiles were investigated in Pseudomonas fluorescens DF57 and Pseudomonas putida DF14 during starvation for carbon, nitrogen, and phosphorus. P. fluorescens DF57 remained fully culturable for 4 days in all starvation regimes. The cell mass increased during starvation for nitrogen and phosphorus, indicating the accumulation of storage compounds, whereas it decreased slightly in carbon-starved cells. P. putida DF14 lost culturability during phosphorus starvation, and the mass of phosphate-starved cells did not increase. Analysis of additional P. fluorescens and P. putida strains, however, showed that the ability to preserve culturability during phosphorus starvation was not species but strain dependent. In DF57, an outer membrane protein of 55 kDa appeared during starvation for phosphorus, while another protein of 63 kDa was seen during all starvation conditions. DF14 induced two outer membrane proteins of 28 and 29 kDa during starvation for carbon and nitrogen, but no phosphorus-specific starvation protein could be detected. Therefore, starvation-induced outer membrane proteins do not seem to be conserved among the fluorescent pseudomonads and a unique starvation response might be found in individual strains.

Survival of Bacillus licheniformis in Seawater Model Ecosystems.

The fate of Bacillus licheniformis DSM 13 was monitored after introduction into laboratory microcosms and mesocosms established in the Knebel Vig estuary, Denmark. The model organism was detected by a combination of immunofluorescence microscopy and nonselective plating followed by colony blotting. This allowed simultaneous quantification of intact cells and culturable cells. B. licheniformis DSM 13 adapted poorly to the conditions in filtered (0.2-mum-pore-size filter) seawater. Results from additional microcosm studies using natural seawater demonstrated that protozoan grazing also was important in regulating the population of the introduced model organism. In experiments using mesocosms, B. licheniformis DSM 13 also showed a rapid die-off. The introduction of the organism led to increased nutrient levels and to increased growth of both autotrophic and heterotrophic components of the plankton community compared with those of control enclosures. Thereby, a more intensive predation impact on the bacterioplankton community was induced. The combination of microcosm and mesocosm experiments provides a scenario in which the influence of single biotic and abiotic factors on survival of introduced organisms can be tested and in which the effect of the introduction on ecosystem structure and function can be evaluated. This test concept might prove useful in risk assessment of genetically modified microorganisms.

Survival of 2,4-dichlorophenoxyacetic acid degrading Alcaligenes eutrophus AE0106(pR0101) in lake water microcosms.

Survival of the 2,4-dichlorophenoxyacetic acid (2,4-D) degrading Alcaligenes eutrophus strain AEO 106 harboring the catabolic plasmid pRO101 was studied in lake water from the eutrophic lake Frederiksborg Slotssø. Survival experiments were performed for periods of 7 days in laboratory microcosms containing filtered (0.2-µm pore size) or natural lake water amended with increasing concentrations of 2,4-D. A. eutrophus AE0106 was detected by combining the fluorescent antibody method with selective and nonselective plating followed by colony blotting and colony hybridization. Comparison of colony blotting and colony hybridization demonstrated that the A. eutrophus AE0106 host organism and the catabolic plasmid pRO101 had similar fates in the model system employed. In all experiments culturable counts of A. eutrophus AE0106 were lower than fluorescent antibody counts and frequently a decline in culturable counts occurred at times when the fluorescent antibody method showed an increasing population size. Amendment with 2,4-D increased survival of A. eutrophus AE0106 both in filtered and in natural lake water. Survival was always poorer in model systems with natural water than in 0.2 µm-filtered water.

Enzyme-linked immunosorbent assays for detection of Pseudomonas fluorescens in sediment samples.

A strain-specific antibody to Pseudomonas fluorescens was used to develop two enzyme-linked immunosorbent assays: a sandwich and a competitive assay. Both assay types could be used to perform rapid and sensitive detection of the target organism in extracts of sediment samples.

A developmental study of soluble L1.

L1 is a neural cell adhesion molecule expressed by neurons and it is involved in cell interactions during axon elongation and fasciculation. L1 from rat brain consists of a membrane-inserted Mr 200,000 polypeptide from which two polypeptides of Mr 180,000 and Mr 140,000 can be derived. These latter polypeptides appear both as membrane-associated and as soluble molecules. In this report, both total and soluble L1 in rat brain have been quantified by an enzyme-linked immunosorbent assay. The amount of total L1 per gram brain varies with postnatal age showing a peak value at postnatal day 7. The variation in soluble L1 coincides with the changes in total L1. Thus, soluble L1 constitutes ca 2% of total L1 at all ages investigated. The soluble Mr 140,000 and 180,000 L1 polypeptides are also present in cerebrospinal fluid. Studies of membrane L1 catabolism in cultured fetal rat brain neurons show that the half-life of membrane L1 is less than 24 hr. As a part of membrane L1 catabolism, small amounts of soluble L1 polypeptides are released to include cell surroundings.