A comparison of enumeration methods for culturable Pseudomonas fluorescens cells marked with green fluorescent protein

The detection of bacteria in environmental samples using genetic markers is valuable in microbial ecology. The green fluorescent protein (GFP) reporter gene was studied under nutrient starvation conditions at 4 degrees C, 23 degrees C and 30 degrees C in Pseudomonas fluorescens R2fG1 cells tagged with a red-shifted gfp. Fluorescence intensity was not significantly different in cells maintained in a buffer for at least 48 days at all the tested temperatures. gfp-Tagged R2fG1 cells were introduced into bulk soil microcosms and soil microcosms with wheat seedlings. GFP-marked cells were enumerated immediately after inoculation into soil and again in soil and root samples after 10 days. Counts of culturable colonies were obtained from drop plates using 5-microl aliquots of serial dilutions viewed with an epifluorescent microscope. Traditional spread plates (using 100-microl aliquots) and the most-probable-number (MPN) method using a spectrofluorometer were also used to enumerate the GFP-marked Pseudomonas cells in soil, rhizosphere and rhizoplane samples. Microcolonies were visualized on root surfaces under the epifluorescent microscope after immobilizing in agar and incubation for 24 h. Counts from traditional spread plates were significantly higher (P<0.05) than the population estimates of the MPN method for all treatments at any sampling time. Counts using the drop plate method, however, were not significantly different (P<0.05) except in one treatment, and provided similar estimates in half the time of spread plates and at an estimated third of the cost.     

Development and application of a most-probable-number-pcr assay to quantify flagellate populations in soil samples

This paper reports on the first successful molecular detection and quantification of soil protozoa. Quantification of heterotrophic flagellates and naked amoebae in soil has traditionally relied on dilution culturing techniques, followed by most-probable-number (MPN) calculations. Such methods are biased by differences in the culturability of soil protozoa and are unable to quantify specific taxonomic groups, and the results are highly dependent on the choice of media and the skills of the microscopists. Successful detection of protozoa in soil by DNA techniques requires (i) the development and validation of DNA extraction and quantification protocols and (ii) the collection of sufficient sequence data to find specific protozoan 18S ribosomal DNA sequences. This paper describes the development of an MPN-PCR assay for detection of the common soil flagellate Heteromita globosa, using primers targeting a 700-bp sequence of the small-subunit rRNA gene. The method was tested by use of gnotobiotic laboratory microcosms with sterile tar-contaminated soil inoculated with the bacterium Pseudomonas putida OUS82 UCB55 as prey. There was satisfactory overall agreement between H. globosa population estimates obtained by the PCR assay and a conventional MPN assay in the three soils tested.     

Survival of lux-lac-marked biosurfactant-producing Pseudomonas aeruginosa UG2L in soil monitored by nonselective plating and PCR.

Two reporter systems, lacZY and luxAB, were stably integrated into the chromosome of Pseudomonas aeruginosa UG2, a biosurfactant-producing strain. Growth and rhamnolipid production of the UG2 wild-type and reporter gene-bearing UG2L strains were similar in liquid culture. A spontaneous rifampin-resistant detecting UG2Lr, allowed antibiotic selection. Phenotypic characteristics were compared for usefulness in detecting UG2Lr colonies: morphology, fluorescent pigment production, light emission (lux), X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) cleavage (lac), and rifampin resistance. Survival patterns of UG2, UG2L, and UG2Lr strains were similar in sandy loam soil microcosms over 12 12 weeks. The lac marker was not suitable for monitoring P. aeruginosa UG2Lr in soil since 20 to 42% of cultured, aerobic, heterotrophic soil microorganisms formed blue, lactose-positive colonies. The lux genes provided a stable and unequivocal reporter system, as effective as conventional antibiotic plating, for tracking microorganisms nonselectively at 10(3) CFU/g of soil. Three months after inoculation into oil-contaminated and uncontaminated soil microcosms, UG2Lr cells were recovered at 10(7) and 10(4) cells per g (dry weight) of soil, respectively. Detection by PCR amplification of part of the luxA gene confirmed a decrease in UG2Lr cell numbers in uncontaminated soil. In combination, antibiotic resistance, bioluminescence, and PCR analyses provided sensitive detection and quantitative enumeration of P. aeruginosa UG2Lr in soil.     

Immunofluorescence studies of Nitrobacter populations in soils.

Certain steps of a protocol to enumerate a bacterium directly in soil by immunofluorescence were studied with respect to the enumeration of Nitrobacter in soils of diverse properties. Maximal counts of Nitrobacter were obtained by varying factors involved in the release of bacteria from the soil. Differences with respect to these factors were related to soil-colloidal properties. Enumeration protocols modified with regard to soil properties were used with strain-specific fluorescent antibodies (FA) to enumerate Nitrobacter populations (a) in soil during storage, (b) in comparison with most probable number (MPN) enumeration, and (c) occurring in a spectrum of soil samples. FA counting was rapid and precise and gave counts generally 10- to 100-fold higher than obtained by MPN. Nitrobacter cells of the two serotypes studied, designated as agilis and Winogradsky, occurred in each soil at levels of 10(4)-10(5) per gram.     

Polychlorinated biphenyl (PCB) degradation and persistence of a<Emphasis Type="Italic"> gfp</Emphasis>-marked<Emphasis Type="Italic"> Ralstonia eutropha</Emphasis> H850 in PCB-contaminated soil

Ralstonia eutropha H850 was labelled chromosomally with a gfp marker gene encoding for the green fluorescent protein, and designated R. eutropha H850g13. Visual observation of green fluorescent cells under an epifluorescence microscope, and PCR amplification products, confirmed that the bacterium was labelled with gfp. Southern blot hybridization products further confirmed the gfp was chromosomally labelled. Using resting cell assays, it was determined that insertion of the gfp gene decreased the microorganisms' ability to degrade biphenyl compared to the parent strain. However, this marker facilitated the identification and monitoring of R. eutropha H850g13 survival in soil microcosm experiments. Survival and polychlorinated biphenyl degradation by R. eutropha H850g13 was analysed in soil microcosms spiked with 2,2,5,5-tetrachlorobiphenyl (TeCB). R. eutropha H850g13 was detected by viable plate counts and most-probable-number/PCR after 102 days in TeCB-contaminated soil microcosms, and was likely outcompeted by indigenous soil microorganisms in microcosms amended with oil and Daramend (an organic amendment, ). R. eutropha H850g13 did not degrade TeCB in any of the soil microcosms. This research confirmed that gfp was useful as a marker to distinguish R. eutropha H850g13 from indigenous soil microorganisms over a 102 day period and that, under the experimental conditions used, R. eutropha H850g13 did not degrade TeCB.     

Development and Application of a Most-Probable-Number–PCR Assay To Quantify Flagellate Populations in Soil Samples

This paper reports on the first successful molecular detection and quantification of soil protozoa. Quantification of heterotrophic flagellates and naked amoebae in soil has traditionally relied on dilution culturing techniques, followed by most-probable-number (MPN) calculations. Such methods are biased by differences in the culturability of soil protozoa and are unable to quantify specific taxonomic groups, and the results are highly dependent on the choice of media and the skills of the microscopists. Successful detection of protozoa in soil by DNA techniques requires (i) the development and validation of DNA extraction and quantification protocols and (ii) the collection of sufficient sequence data to find specific protozoan 18S ribosomal DNA sequences. This paper describes the development of an MPN-PCR assay for detection of the common soil flagellate Heteromita globosa, using primers targeting a 700-bp sequence of the small-subunit rRNA gene. The method was tested by use of gnotobiotic laboratory microcosms with sterile tar-contaminated soil inoculated with the bacterium Pseudomonas putida OUS82 UCB55 as prey. There was satisfactory overall agreement between H. globosa population estimates obtained by the PCR assay and a conventional MPN assay in the three soils tested.     

Intracellular detection assays for high-throughput screening

New optical assay methods promise to accelerate the use of living cells in screens for drug discovery. Most of these methods employ either fluorescent or luminescent read-outs and allow cell-based assays for most targets, including receptors, ion channels and intracellular enzymes. Furthermore, genetically encoded probes offer the possibility of custom-engineered biosensors for intracellular biochemistry, specifically localized targets, and protein—protein interactions.     

A flow cytometry-optimized assay using an SOS-green fluorescent protein (SOS-GFP) whole-cell biosensor for the detection of genotoxins in complex environments

Whole-cell biosensors have become popular tools for detection of ecotoxic compounds in environmental samples. We have developed an assay optimized for flow cytometry with detection of genotoxic compounds in mind. The assay features extended pre-incubation and a cell density of only 10(6)-10(7) cells/mL, and proved far more sensitive than a previously published assay using the same biosensor strain. By applying the SOS-green fluorescent protein (GFP) whole-cell biosensor directly to soil microcosms we were also able to evaluate both the applicability and sensitivity of a biosensor based on SOS-induction in whole soil samples. Soil microcosms were spiked with a dilution-series of crude broth extract from the mitomycin C-producing streptomycete Streptomyces caespitosus. Biosensors extracted from these microcosms after 1 day of incubation at 30 degrees C were easily distinguished from extracts of non-contaminated soil particles when using flow cytometry, and induction of the biosensor by mitomycin C was detectable at concentrations as low as 2.5 ng/g of soil.     

Enumerating low densities of genetically engineered Erwinia carotovora in soil

An inexpensive, quantitative, and sensitive technique was developed for detection of genetically engineered Erwinia carotovora in soil samples. Enrichment media, antibiotic resistance, and most probable number (MPN) analysis were used to enumerate as few as 1 to 10 target cells/10 g soil. The MPN technique recovered significantly higher cell densities than plating; however, densities estimated by the two techniques were strongly correlated. After inoculation of soil microcosms with genetically engineered E. carotovora, a decline rate of 1.2 log units/g soil/10 days and then subsequent disappearance was observed using the MPN technique.     

Survival of free and alginate-encapsulated Pseudomonas aeruginosa UG2Lr in soil treated with disinfectants

Pseudomonas aeruginosa UG2Lr, a rifampicin-resistant strain possessing the luxAB on a chromosomal Tn5 insert, was inoculated into soil microcosms as either free cells or encapsulated in dry alginate beads. A 100-fold increase in cell number g^-1 dry soil was observed in microcosms inoculated with alginate-encapsulated UG2Lr after 3 weeks incubation at 22°C compared to microcosms inoculated with free cells. After 98 d, microcosms inoculated with free UG2Lr cells contained 10⁴ cfu g^-1 dry soil compared to 10⁷ cfu g^-1 dry soil in microcosms inoculated with alginate-encapsulated UG2Lr cells. The effects of disinfectants on both the free and alginate-encapsulated UG2Lr cells were also examined. 1·0% (w/g dry soil) calcium hypochlorite, formaldehyde and Spectrum Clear Bath, were added to microcosms each week for 4 weeks. Formaldehyde killed both free and alginate-encapsulated UG2Lr cells within 14 d after only two amendments. Calcium hypochlorite reduced free UG2Lr cell numbers 10-fold 2 d after initial application; however, the introduced population recovered and was unaffected by subsequent treatments at 7, 14 and 21 d. Alginate-encapsulated UG2Lr cells were not affected by calcium hypochlorite treatment. Spectrum Clear Bath did not kill either free or alginate-encapsulated UG2Lr cells in soil. Alginate encapsulation improved survival of introduced bacteria in soil except in the presence of formaldehyde. Killing genetically-engineered bacteria in soil may be difficult unless a powerful disinfectant such as formaldehyde is used or the genetically-engineered micro-organism is allowed to become non-viable over time.     

Nitrification and denitrification in soil: A comparison of enzyme assay,incubation and enumeration methods

Summary Three methods for assaying nitrification and denitrification were compared in agricultural field plots in the Southeastern USA. Nitrification was measured using the chlorate inhibition enzyme assay, by measuring (NO ₃ ⁻ −N) production in controlled incubations and by most probable number (MPN) determinations. Denitrification was measured by the phase I enzyme assay, by incubation of soil cores and by MPN determinations. The methods were compared in terms of their representation of seasonal patterns and treatment differences. The enzyme assays were most effective for showing treatment differences because they measure maximum potential enzyme biomass activity which is an integrated product of treatment effects. The incubation methods required minimal alteration ofin situ soil conditions but were confounded by other biological processes and by high spatial and temporal variabiltiy. MPN determinations were time consuming and were least effective for illustrating treatment differences and seasonal patterns.     

Use of a commercial gene probe assay kit for rapid MPN enumeration of Escherichia coli in drinking water

A commercial gene probe assay kit for presence/absence determination of Escherichia coli in food samples has been used in the standard UK six tube format most probable number (MPN) method for enumerating E. coli in drinking water samples. Presence/absence analysis with the gene probe kit (requiring 3 h) of all MPN tubes after a 21–24 h incubation (minerals modified glutamate; 37°C) enumerated confirmed E. coli in 24–27 h which offered an improvement of up to 48 h over the standard UK MPN method. MPNs determined by the gene probe method and the standard UK method agreed in nine of the 16 water samples which were analysed and for which E. coli concentrations were within the detection limits of the six tube MPN format. This was consistent with the gene probe method detecting one E. coli in a tube. For the other seven water samples, the gene probe method registered positive only 20 of the 30 tubes which the standard UK method determined to be positive. The sensitivity of the gene probe method for drinking water samples, although encouraging, needs improvement perhaps through kit quality control procedures.     

Fate of Escherichia coli O157:H7 in irrigation water on soils and plants as validated by culture method and real-time PCR

One of the most common vehicles by which Escherichia coli O157:H7 may be introduced into crops is contaminated irrigation water. Water contamination is becoming more common in rural areas of the United States as a result of large animal operations, and up to 40% of tested drinking-water wells are contaminated with E. coli. In this study, 2 contrasting soil samples were inoculated with E. coli O157:H7 expressing green fluorescent protein through irrigation water. Real-time PCR and culture methods were used to quantify the fate of this pathogen in phyllosphere (leaf surface), rhizosphere (volume of soil tightly held by plant roots), and non-rhizosphere soils. A real-time PCR assay was designed with the eae gene of E. coli O157:H7. The probe was incorporated into real-time PCR containing DNA extracted from the phyllosphere, rhizosphere, and non-rhizosphere soils. The detection limit for E. coli O157:H7 quantification by real-time PCR was 1.2 x 10(3) in the rhizosphere, phyllosphere, and non-rhizosphere samples. E. coli O157:H7 concentrations were higher in the rhizosphere than in the non-rhizosphere soils and leaf surfaces, and persisted longer in clay soil. The persistence of E. coli O157:H7 in phyllosphere, rhizosphere, and non-rhizosphere soils over 45 days may play a significant part in the recontamination cycle of produce in the environment. Therefore, the rapidity of the real-time PCR assay may be a useful tool for quantification and monitoring of E. coli O157:H7 in irrigation water and on contaminated fresh produce.     

Detection and counting of Nitrobacter populations in soil by PCR.

Although the biological conversion of nitrite to nitrate is a well-known process, studies of Nitrobacter populations are hindered by their physiological characteristics. This report describes a new method for detecting and counting Nitrobacter populations in situ with the PCR. Two primers from the 16S rRNA gene were used to generate a 397-bp fragment by amplification of Nitrobacter species DNA. No signal was detected from their phylogenetic neighbors or the common soil bacteria tested. Extraction and purification steps were optimized for minimal loss and maximal purity of soil DNA. The detection threshold and accuracy of the molecular method were determined from soil inoculated with 10, 10(2), or 10(3) Nitrobacter hamburgensis cells per g of soil. Counts were also done by the most-probable-number (MPN)-Griess and fluorescent antibody methods. PCR had a lower detection threshold (10(2) Nitrobacter cells per g of soil) than did the MPN-Griess or fluorescent antibody method. When PCR amplification was coupled with the MPN method, the counting rate reached 65 to 72% of inoculated Nitrobacter cells. Tested on nonsterile soil, this rapid procedure was proved efficient.     

N-glycosylation is required for efficient secretion of a novel human secreted glycoprotein, hPAP21

The present study reported the isolation and characterization of a novel human secreted protein, named as hPAP21 (human protease-associated domain-containing protein, 21 kDa), encoded by the hypothetical gene chromosome 2 open reading frame 7 (C2orf7) that contains signal peptide in its N-terminus, without transmembrane domain, except for PA domain in its middle. Western blotting assay indicated that the c-Myc tagged hPAP21 could be secreted into culture medium in the transfected Chinese hamster ovary cells. However, the molecular weights, whatever intracellular (28 kDa) or extracellular (30 kDa) forms, are larger than that of the prediction. To define whether the glycosylation was important process for its secretion, endoglycosidase H (Endo H) and PNGase F (PNG F) were employed to evaluate the effect of glycosylation types on secretion of hPAP21. Interestingly, the extracellular forms were primarily sensitive to PNG F, not Endo H, implying that complex N-glycosylation could be required for the secretion of hPAP21. Furthermore, N-glycosylation of Asn171 was confirmed as potential crucial process for the secretory protein via site-directed mutagenesis assay. All data will be contributed to the understanding of molecular functions of hPAP21.     

An indirect fluorescent antibody staining technique for determining population levels ofThiobacillus ferrooxidans in acid mine drainage waters

Thiobacillus ferrooxidans is believed to be responsible for the oxidation of ferrous ion at low pH, the rate-limiting step in the oxidation of pyrite ores and subsequent formation of acid mine drainage (AMD). It has been suggested that efforts to control this environmental problem include procedures that would inhibit this bacterium. At present, a most probable number (MPN) procedure requiring a minimum of 10 days is used to enumerate this microorganism in natural waters. If control of AMD through inhibition ofT. ferrooxidans is to be feasible, it will be necessary to develop a more rapid method to determine population levels to facilitate application of control measures.An indirect fluorescent antibody (FA) staining technique was developed for this purpose which provided reliable estimates within a few hours. Artificial samples containing approximated numbers ofT. ferrooxidans were analyzed using the FA and MPN procedures, and the FA technique more closely approximated expected numbers of cells. The MPN method was excessively conservative, detecting only 3% to 21% of the cells enumerated by the FA procedure.     

The effectiveness of molecular markers for the identification of Lr28, Lr35, and Lr47 genes in common wheat

The effectiveness of molecular markers for the identification of leaf rust resistance genes Lr28, Lr35 and Lr47 transferred to common wheat from Ae. speltoides was assessed using samples of Triticum spp. and Aegilops spp. The markers Sr39F2/R3, BCD260F1/35R2 of the gene Lr35 and PS10 of the Lr47 gene were characterized by high efficiency and were revealed in the lines of common wheat containing these genes, and samples of Ae. speltoides species, the donor of these genes. The marker SCS421 of the Lr28 gene and the markers Sr39#22r, Sr39#50s, BE500705 of the Lr35/Sr39 genes turned out to be less specific. The marker SCS421 was amplified in the samples of the T. timopheevii species, line KS90WRC010 (Lr41), the cultivar of common wheat Pamyati Maystrenko, obtained using synthetic hexaploid T. timopheevii × Ae. tauschii and introgressive lines obtained using Ae. speltoides. The marker BE500705, which indicates the absence of the Lr35/Sr39 genes, was not revealed in the lines TcLr35 and MqSr39, in Ae. speltoides, Ae. tauschii and T. boeoticum (kk-61034, 61038). Analysis of the nucleotide sequences of amplification products obtained with the markers SCS421 and Sr39#22r indicated their low homology with TcLr28 and TcLr35. Using molecular markers, a different distribution of the Lr28 (77%), Lr35 (100%) and Lr47 (15%) genes in 13 studied samples of Ae. speltoides was shown. In introgressive lines derived from Ae. speltoides, contemporary Russian cultivars of common wheat and triticale the Lr28, Lr35, Lr47 genes were not revealed.     

A fluorescent alpha-factor analogue exhibits multiple steps on binding to its G protein coupled receptor in yeast

The yeast alpha-factor receptor encoded by the STE2 gene is a member of the extended family of G protein coupled receptors (GPCRs) involved in a wide variety of signal transduction pathways. We report here the use of a fluorescent alpha-factor analogue [K(7)(NBD), Nle(12)] alpha-factor (Lys(7) (7-nitrobenz-2-oxa-1,3-diazol-4-yl), norleucine(12) alpha-factor) in conjunction with flow cytometry and fluorescence microscopy to study binding of ligand to the receptor. Internalization of the fluorescent ligand following receptor binding can be monitored by fluorescence microscopy. The use of flow cytometry to detect binding of the fluorescent ligand to intact yeast cells provides a sensitive and reproducible assay that can be conducted at low cell densities and is relatively insensitive to fluorescence of unbound and nonspecifically bound ligand. Using this assay, we determined that some receptor alleles expressed in cells lacking the G protein alpha subunit exhibit a higher equilibrium binding affinity for ligand than the same alleles expressed in isogenic cells containing the normal complement of G protein subunits. On the basis of time-dependent changes in the intensity and shape of the emission spectrum of [K(7)(NBD),Nle(12)] alpha-factor during binding, we infer that the ligand associates with receptors via a two-step process involving an initial interaction that places the fluorophore in a hydrophobic environment, followed by a conversion to a state in which the fluorophore moves to a more polar environment.