Biomedical subjects

S J Sørensen

Publications and source records attributed to S J Sørensen.

16 recordsLinked to original sources

PubMed2004

Recovery of GFP-labeled bacteria for culturing and molecular analysis after cell sorting using a benchtop flow cytometer.

Exciting opportunities exist for the application of simple fluorescence-activated cell sorting (FACS) to microbiology. The technology is widely available, but critical reports on the efficiency of cell sorting using benchtop instruments are lacking. It is vital that single cell sorting be of the highest purity possible. If purity is compromised detrital material or unwanted cells will be captured along with target cells of interest. Here, the isolation of fluorescent bacteria using a benchtop FACSCalibur-sort flow cytometer is described. The efficiency and purity of isolated cells was determined using fluorescence microscopy, culturing, and molecular analysis. To achieve high purity it was essential that the total event rate did not exceed 300 cells per second. This instrument was capable of recovering >55% sorted Escherichia coli cells, coupled with a purity exceeding 99%. However, the purity of recovered cells was substantially reduced (<25%) when the event rate increased. Cell sorting onto polycarbonate membranes did not reduce the ability of E. coli to form colonies, and sorting of ~1000 E. coli cells was sufficient for 16S rDNA amplification. Additionally, as few as 100 isolated Erwinia sp. carrying the gfp gene were amplified using seminested PCR targeting the single copy gfp gene. With such low numbers of bacteria being required for molecular identification, FACS can be achieved without the requirement for high-speed droplet cell sorters.

Cell Separation↗

PubMed2003

Presence of N-acyl homoserine lactones in soil detected by a whole-cell biosensor and flow cytometry.

Quorum sensing enables bacteria to regulate expression of certain genes according to population density. N-acyl homoserine lactone (AHL)-based quorum sensing is known to be widespread among gram-negative bacteria. Several bacterial whole-cell biosensors for AHL detection have been developed and some were used in in situ studies of AHL production. From these studies our knowledge of the significance of quorum sensing in various environments has been improved. However, very little is known about production of AHLs in soil environments. In the present study, an approach for detecting AHL production in bulk soil was developed. A whole-cell biosensor based on the regulatory region of the lux-operon from Vibrio fischeri fused to gfp was constructed, resulting in a luxR-PluxI-gfpmut3*-fusion in the high copy plasmid, pAHL-GFP. Escherichia coli MC4100 harboring pAHL-GFP responded to the AHL-compound N-octanoyl homoserine lactone (OHL) by expressing green fluorescence. In situ application of E. coli MC4100/pAHL-GFP was tested by adding OHL in different concentrations to sterile soil microcosms. E. coli MC4100/pAHL-GFP were incubated in the soil microcosms and extracted by an improved Nycodenz-extraction method optimized for flow cytometry. The presence of induced cells was then verified by single-cell analysis by flow cytometry. OHL concentrations between 0.5 and 50 nmol per g soil were detected. When introducing the AHL-producing Serratia liquefaciens to soil microcosms, expression of green fluorescent protein was induced in E. coli MC4100/pAHL-GFP. Thereby, the ability of this strain to detect excretion of AHLs by S. liquefaciens in sterile soil was shown. The use of an improved extraction method and a whole-cell biosensor combined with flow cytometry analysis proved to be promising tools in future studies of AHL production by microbial populations in soil environments.

4-Butyrolactone↗

PubMed2002

The diversity and function of soil microbial communities exposed to different disturbances.

To improve understanding of the relationship between the diversity and function of the soil ecosystem, we investigated the effect of two different disturbances on soil bacterial communities -- long-term exposure to the heavy metal mercury and transient exposure to the antibiotic tylosin. In the mercury-contaminated soil the diversity (Shannon index) was reduced as assessed from denaturing gradient gel electrophoresis (DGGE) of amplified 16S rDNA sequences from the soil community DNA and from colony morphology typing of the culturable bacterial population. However, analysis of the substrate utilization profiles did not reveal any differences in diversity. In the tylosin-treated soil, DGGE revealed a small difference in the diversity of 16S rDNA compared to the control soil, whereas analysis of the colony morphology typing or substrate utilization results did not reveal any differences in diversity. Soil function was also affected by mercury contamination. The lag time before soil respiration increased following addition of glucose or alfalfa substrate was longer in the mercury-contaminated soil than in the control soil. Moreover, it was markedly prolonged in mercury-contaminated soil subjected to heat treatment prior to substrate addition, thus indicating reduced resistance to a new disturbance in the mercury-contaminated soil as compared to the control soil. Tylosin treatment did not have any significant effect on any of the respiration parameters measured, either with or without prior heat treatment of the soil.

Bacteria↗

PubMed2001

Adaptation of the bacterial community to mercury contamination.

The utilisation of 31 sole carbon sources by bacterial communities of soil in the presence of increasing concentrations of Hg(II) was measured by a colour development assay. The assay was performed on Biolog microtitre plates (Ecoplates) in the presence of Hg(II) and compared to Hg(II)-free Ecoplates. Furthermore, community tolerance to Hg(II) was measured by colour development in microtitre plates supplemented with LB broth and by enumeration of colony-forming units on LB agar plates. Both microtitre plates supplemented with LB and LB agar plates contained increasing concentrations of Hg(II). The difference in substrate utilisation profile, as shown by growth on 31 different carbon substrates in the Ecoplates, suggested an adaptation of the soil community that correlated with the metal exposure level in the soil. Similarly, growth on microtitre plates supplemented with LB and plate-spreading data showed an increased community tolerance with increasing levels of mercury in the soil. Both the multi-function microtitre plate assay (Ecoplate) and the LB broth microtitre plate assay are suitable for evaluating the adaptation of the bacterial community in soil to a heavy metal pollutant.

Adaptation, Physiological↗

PubMed2001

Effect of genomic location on horizontal transfer of a recombinant gene cassette between Pseudomonas strains in the rhizosphere and spermosphere of barley seedlings.

The use of genetically engineered bacteria in natural environments constitutes a risk of transfer of recombinant DNA to the indigenous bacteria. However, chromosomal genes are believed to be less likely to transfer than genes on mobilizable and conjugative plasmids. To study this assumption, horizontal transfer of a recombinant gene cassette inserted into the chromosome of a Pseudomonas stutzeri strain, into a mobilizable plasmid (pAGM42), and into a conjugative plasmid (pKJK5) isolated from barley rhizosphere was investigated. Horizontal transfer efficiencies of the gene cassette inserted into a conjugative plasmid was 8.20 x 10(-3) transconjugants/(donors x recipients)1/2 in the rhizosphere and 4.57 x 10(-2) transconjugants/(donors x recipients)1/2 in the spermosphere. Mobilization of the plasmid pAGM42 by the plasmids RP4 and pKJK5 was also detected at high levels in the microcosms, transfer efficiencies were up to 4.36 x 10(-3) transconjugants/(donors x recipients)1/2. Transfer of chromosomal encoded genes could not be detected in the microcosms by conjugation or transformation. However, transformation did occur by using the same bacterial strains under laboratory conditions. The rhizosphere and especially the spermosphere thus proved to be hot spot environments providing favorable conditions for gene transfer by mobilization and conjugation, but these environments did not support transformation at a detectable level.

Chromosomes, Bacterial↗

PubMed2001

Detection of oxytetracycline production by Streptomyces rimosus in soil microcosms by combining whole-cell biosensors and flow cytometry.

Combining the high specificity of bacterial biosensors and the resolution power of fluorescence-activated cell sorting (FACS) provided qualitative detection of oxytetracycline production by Streptomyces rimosus in soil microcosms. A plasmid containing a transcriptional fusion between the tetR-regulated P(tet) promoter from Tn10 and a FACS-optimized gfp gene was constructed. When harbored by Escherichia coli, this plasmid produces large amounts of green fluorescent protein (GFP) in the presence of tetracycline. This tetracycline biosensor was used to detect the production of oxytetracycline by S. rimosus introduced into sterile soil. The tetracycline-induced GFP-producing biosensors were detected by FACS analysis, enabling the detection of oxytetracycline encounters by single biosensor cells. This approach can be used to study interactions between antibiotic producers and their target organisms in soil.

Biosensing Techniques↗

PubMed2000

Versatile biosensor vectors for detection and quantification of mercury.

Three different whole cell biosensor constructs were made by fusing the mercury inducible promoter, P(mer), and its regulatory gene, merR, from transposon Tn21 with reporter genes luxCDABE, lacZYA, or gfp. In Escherichia coli these biosensor constructs responded to low levels of mercury by producing light, beta-galactosidase or green fluorescent protein, respectively. Since the responses were quantitative, the constructs were used to quantify bioavailable mercury in different environments. The constructs were cloned into mini-Tn5 delivery vectors, thus enabling the transfer of the mer-lux, mer-lac or mer-gfp cassettes to a variety of Gram-negative bacteria. The mer-lux cassette was transferred to a Pseudomonas putida strain, which was used to quantify water-extractable mercury in contaminated soil.

Bacterial Proteins↗

PubMed2000

Detection and quantification of tetracyclines by whole cell biosensors.

Three different mini-Tn5 plasmids, containing a tetracycline-inducible promoter, Ptet and a regulatory gene, tetR, in operon fusions with a reporter gene system (lacZYA, luxCDABE or gfp), were constructed. These biosensor constructs responded to low levels of tetracyclines by producing beta-galactosidase, light or green fluorescent protein. They did so in a quantitative manner, thus enabling the quantification of tetracyclines in the immediate surroundings of the biosensor organism. All three constructs were transferred successfully to different gram-negative bacteria by conjugation. An Escherichia coli strain containing the Ptet-lac construct was used to determine oxytetracycline in milk as a demonstration of the application of these biosensors.

Animals↗

PubMed1999

Utilization of phenoxyacetic acid, by strains using either the ortho or meta cleavage of catechol during phenol degradation, after conjugal transfer of tfdA, the gene encoding a 2,4-dichlorophenoxyacetic acid/2-oxoglutarate dioxygenase.

The degradation of recalcitrant pollutants in contaminated soils and waters could be facilitated by broadening the degradative capabilities of indigenous microbes by the conjugal transfer of catabolic genes. The feasibility of establishing bacterial populations that degrade phenoxyacetic acid by conjugal transfer of tfdA, the gene encoding 2,4-dichlorophenoxyacetic acid/2-oxoglutarate dioxygenase, to phenol-degrading strains of Pseudomonas and Ralstonia was examined. The mobilizable plasmid pKJS32 served as a vector for delivery of tfdA and the regulatory gene, tfdS. Transconjugant strains that degraded phenol by an ortho cleavage of catechol grew well on phenoxyacetic acid while those employing a meta cleavage could only grow on phenoxyacetic acid in the presence of benzoic acid or after a prolonged lag period and the appearance of mutants that had gained catechol 1,2-dioxygenase activities. Thus, an ortho cleavage of catechol was essential for degradation of phenoxyacetic acid, suggesting that a product of the ortho-cleavage pathway, probably cis, cis-muconic acid, is an inducer of tfdA gene expression. Establishment of phenoxyacetic-acid-degrading soil populations by conjugal transfer of tfdA would depend on the presence of phenol-degrading recipients employing an ortho cleavage of catechol.

2,4-Dichlorophenoxyacetic Acid↗

PubMed1998

The effect of longterm exposure to mercury on the bacterial community in marine sediment.

The aim of this study was to investigate the effect of mercury contamination on bacterial community structure and function. Bacterial communities from two sites, a mercury-contaminated site inside the harbor of Copenhagen, Denmark (CH) and a unpolluted control site, Koge Buge (KB), were compared with respect to diversity indices, of antibiotic- and heavy metal-resistance patterns, abundance and self transmissibility of plasmids in resistant isolates (endogenous isolation). Furthermore, the potential for gene transfer between indigenous bacteria was assessed by the exogenous plasmid isolation approach. It was found that resistance to all the tested compounds was higher in the mercury-polluted sediment than the control sediment. The abundance of plasmids was higher at the polluted site, where 62% of the isolates contained plasmids, whereas only 29% of the isolates from the control sediment contained plasmids. Furthermore, the frequencies of large plasmids and plasmids per isolates were found to be higher in the contaminated sediment. Exogenous plasmid isolations revealed high occurrence of Hg and tetracycline resistance, self-transmissible plasmids in CH sediment (1.8 x 10(-5) transconjugants per recipients) relative to KB sediment (3.0 x 10(-8) T/R). Shannon-Weaver diversity indices showed no difference in the diversity of the isolates from the two sites, and Hg-resistant isolates from CH were found to be as diverse as the CH isolates in total. This may be owing to high level of self-transmissible Hg resistance plasmids found in CH.

Bacteria↗

PubMed1998

The effect of the lacY gene on the induction of IPTG inducible promoters, studied in Escherichia coli and Pseudomonas fluorescens.

The role of the Escherichia coli lacY gene product (the lactose permease) in the induction of isopropyl-beta-D-thiogalactopyranoside (IPTG) inducible promoters was studied in E. coli and P. fluorescens. This was done by comparing strains containing a lacIPOZYA chromosomal insert with newly constructed strains containing inserts without the lacY gene (lacIPOZ). The lactose operon inserts were introduced as single-copy chromosomal inserts to eliminate differences in expression caused by differences in copy number. Comparison between the two types of inserts showed that the lactose permease was essential to allow growth on lactose by both bacteria and that the lactose permease plays an important role in transporting the inducer IPTG across the membrane of P. fluorescens. The use of a functional lactose permease allows expression of beta-galactosidase to increase more than fivefold from a wild-type lac promoter in P. fluorescens SS1001. We suggest that an increase in the rate of protein synthesis from lac-type promoters could be enhanced if an active lactose permease is present as well.

Escherichia coli↗

PubMed1998

Methods for detection of conjugative plasmid transfer in aquatic environments.

Donor and recipient counter selection was evaluated by selecting bacteria that received plasmid RP4 by conjugation on filters and in lake water microcosms. Three counter selection systems were compared; (i) Use of antibiotic-resistant recipients, (ii) use of an auxotrophic donor, and (iii) use of a donor with chromosomal suicide genes. Transfer efficiencies of transconjugants per recipient obtained with the three different counter selection systems in filter-matings were not significantly different. Some nalidixic acid-resistant recipients became partly sensitive to nalidixic acid after receiving the plasmid. Use of an auxotrophic donor was a feasible and easy way to recover indigenous transconjugants. A strain with two copies of the suicide gene gef was successfully eliminated in filter-matings, but elimination of the donor in microcosms by induction of the suicide genes did not succeed. Thus, this counter selection system was not usable in microcosm experiments.

Bacteria↗

PubMed1998

Transfer of plasmid RP4 in the spermosphere and rhizosphere of barley seedling.

Transfer of plasmid RP4 to indigenous bacteria in bulk soil could only be detected in soil with nutrient amendment. Lack of physiological active donor and recipient cells was apparently one of the limiting factors in un-amended bulk soil. Plasmid transfer was detected both in the spermosphere and rhizosphere of barley seedlings. Transfer occurred from seed coated donor bacteria (i) to introduced recipient bacteria and (ii) to indigenous bacteria present in soil. Plasmid transfer was also detected from donor bacteria introduced to the soil to seed coated recipient bacteria. Transfer efficiencies in the rhizosphere were significantly below the transfer efficiencies obtained in the spermosphere. The transfer efficiencies detected in the barley spermosphere were among the highest reported from any natural environment.

Conjugation, Genetic↗

PubMed1997

Chromosomal insertion of the entire Escherichia coli lactose operon, into two strains of Pseudomonas, using a modified mini-Tn5 delivery system.

A 12-kb PstI fragment including the entire E. coli lactose operon (lacIPOZYA) was inserted in one copy into the chromosome of Pseudomonas putida, Pseudomonas fluorescens and an E. coli strain with lac- phenotype. This was made possible by improvements of an already existing mini-Tn5 transposon delivery system (de Lorenzo et al., 1990; Herrero et al., 1990), which integrates cloned DNA fragments at random sites on the chromosome of the recipient bacteria in single copies. This has resulted in: (a) the making of two useful low copy-number cloning vectors both with extensive multi-cloning regions flanked by NotI sites needed in the mini-Tn5 delivery system; (b) the generation of E. coli nonlysogenic strains expressing the pi protein thus being capable of maintaining and delivering R6K-based mini-Tn5 vectors to other E. coli strains; (c) the successful insertion of the E. coli lactose operon into the P. fluorescens chromosome giving P. fluorescens the ability to grow on lactose; (d) evidence from Southern blotting that contradicts the assumption that the mini-Tn5 delivery system always creates one-copy inserts. These improvements allow insertion of large DNA fragments encoding highly expressed proteins into the chromosome of a large variety of Gram-negative bacteria including E. coli.

Chromosomes, Bacterial↗

PubMed1993

Transfer of plasmid RP4 from Escherichia coli K-12 to indigenous bacteria of seawater.

Transfer of plasmid RP4 from Escherichia coli K-12 donor strain to bacteria isolated from seawater was shown to occur on filters and in sterile seawater incubated at 24 degrees C. Ten of 12 seawater isolates tested were recipient active for RP4 when the plasmid transfers were assessed by filter matings. When matings were performed in sterile seawater, seven of the 12 isolates received RP4. In sterile seawater, the transfer of RP4 from E. coli to pseudomonads was more efficient than transfer between E. coli strains. Transfer of RP4 to indigenous seawater bacteria was shown to take place both on filters and in seawater amended with Luria Bertani broth. No transconjugants were found in experiments with unamended seawater, but in experiments with amended seawater 10(-7)-10(-6) transconjugants per recipient were found. In filter matings with a 100-fold-concentrated total population of indigenous seawater bacteria, the transfer efficiency of RP4 was 4 x 10(-5) per recipient. The majority of the isolated transconjugant seawater bacteria consisted of fluorescent pseudomonads, but transconjugant strains of Aeromonas hydrophila, Pseudomonas cepacia, and Enterobacter cloacae were also found. Using an auxotrophic donor strain, selection of transconjugants on selective minimal media was shown to be an efficient strategy for detection of gene transfer to indigenous bacteria of seawater.

Conjugation, Genetic↗

PubMed1992

Mobilization of nonconjugative pBR322-derivative plasmids from laboratory strains of Escherichia coli to bacteria isolated from seawater.

Mobilization of derivatives of plasmids pBR322 and pBR325 was shown to occur between Escherichia coli K12 strains in LB-broth at 37 degrees C, provided a mobilizer plasmid (F') was present either together with the nonconjugative plasmid or in a second donor strain. Evidence from restriction endonuclease analysis suggested that the mobilization was facilitated by a transposition phenomenon involving the "gamma-delta" sequence of F'. It was shown that mobilization of a derivative of pBR325 from E. coli K12 to bacteria isolated from seawater occurred in incubations in both LB-broth and filtered seawater and that Pseudomonas sp., Enterobacter aerogenes, Klebsiella oxytoca, E. coli, and Citrobacter amalonaticus isolates were recipient-active.

Citrobacter↗