A note on the use of a plasmid as a DNA probe in the detection of a Lactobacillus fermentum strain in porcine stomach contents

A plasmid (about 50 kb) was used as a DNA probe to enumerate, by colony hybridization, a strain of Lactobacillus fermentum in the stomach contents of eight piglets. The population sizes obtained by colony hybridization were in agreement with estimated levels calculated on the basis of plasmid profiling of colonies isolated at random from the total lactobacillus population.     

Application of DNA-DNA colony hybridization to the detection of catabolic genotypes in environmental samples

The application of preexisting DNA hybridization techniques was investigated for potential in determining populations of specific gene sequences in environmental samples. Cross-hybridizations among two degradative plasmids, TOL and NAH, and two cloning vehicles, pLAFR1 and RSF1010, were determined. The detection limits for the TOL plasmid against a nonhomologous plasmid-bearing bacterial background was ascertained. The colony hybridization technique allowed detection of one colony containing TOL plasmid among 10(6) Escherichia coli colonies of nonhomologous DNA. Comparisons between population estimates derived from growth on selective substrates and from hybridizations were examined. Findings indicated that standard sole carbon source enumeration procedures for degradative populations lead to overestimations due to nonspecific growth of other bacteria on the microcontaminant carbon sources present in the media. Population estimates based on the selective growth of a microcosm population on two aromatic substrates (toluene and naphthalene) and estimates derived from DNA-DNA colony hybridizations, using the TOL or NAH plasmid as a probe, corresponded with estimates of substrate mineralization rates and past exposure to environmental contaminants. The applications of such techniques are hoped to eventually allow enumeration of any specific gene sequences in the environment, including both anabolic and catabolic genes. In addition, this procedure should prove useful in monitoring recombinant DNA clones released into environmental situations.     

Application of DNA-DNA colony hybridization to the detection of catabolic genotypes in environmental samples.

The application of preexisting DNA hybridization techniques was investigated for potential in determining populations of specific gene sequences in environmental samples. Cross-hybridizations among two degradative plasmids, TOL and NAH, and two cloning vehicles, pLAFR1 and RSF1010, were determined. The detection limits for the TOL plasmid against a nonhomologous plasmid-bearing bacterial background was ascertained. The colony hybridization technique allowed detection of one colony containing TOL plasmid among 10(6) Escherichia coli colonies of nonhomologous DNA. Comparisons between population estimates derived from growth on selective substrates and from hybridizations were examined. Findings indicated that standard sole carbon source enumeration procedures for degradative populations lead to overestimations due to nonspecific growth of other bacteria on the microcontaminant carbon sources present in the media. Population estimates based on the selective growth of a microcosm population on two aromatic substrates (toluene and naphthalene) and estimates derived from DNA-DNA colony hybridizations, using the TOL or NAH plasmid as a probe, corresponded with estimates of substrate mineralization rates and past exposure to environmental contaminants. The applications of such techniques are hoped to eventually allow enumeration of any specific gene sequences in the environment, including both anabolic and catabolic genes. In addition, this procedure should prove useful in monitoring recombinant DNA clones released into environmental situations.     

A specific marker,pat, for studying the fate of introduced bacteria and their DNA in soil using a combination of detection techniques

A specific eucaryotic DNA marker from Solanum tuberosum cv Bintje (688 bp patatin cDNA fragment) was cloned into the unique HindIII-site of plasmid RP4. RP4:: pat was transferred from Escherichia coli to Pseudomonas fluorescens R2f by filter mating.Homology to pat was not detected in the microbial population of Ede loamy sand soil, nor in that of the rhizosphere of wheat growing in this soil, as evidenced by colony filter hybridization. More sensitive molecular detection techniques like most-probable-number recovery/hybridization analysis, and analysis of total community DNA from soil by polymerase chain reaction (PCR) amplification did not reveal the presence of the pat sequence either. P. fluorescens R2f (RP4:: pat), introduced into sterile soil extract microcosms, initially showed poor survival and plasmid loss, after which the introduced populations grew and stabilized at a level of about Log₁₀ 7 cfu per mL. Between 25 and 50% of the population maintained the plasmid, as evidenced by filter hybridization of colonies from non-selective agar plates using the pat fragment as probe.Introduced R2f (RP4:: pat) could be recovered from soil microcosms using selective plating followed by colony hybridization and MPN recovery/hybridization with the pat probe. The presence of the pat marker always coincided with the presence of the resistance genes on RP4:: pat, indicating pat was an adequate marker of the presence of this plasmid. In addition, it adequately described the population dynamics of the introduced strain in soil, since no loss of the plasmid occurred.Hybridization to pat was also useful to show transfer of plasmid RP4:: pat to a recipient strain in soil; transfer to indigenous bacteria was not detected.Analysis by slot-blot hybridization of total community DNA extracted from inoculated soils indicated about Log₁₀ 6 cfu per g of dry soil were still detectable. Application of the PCR on this DNA indicated pat was detectable at least at a level of Log₁₀ 4 immunofluorescence-detectable cells per g of dry soil. Thus extraction of total community DNA followed by PCR permitted the detection of genetically engineered microorganisms present in soil as non-culturable cells.     

Derivation of DNA probes for enumeration of a specific strain of Lactobacillus acidophilus in piglet digestive tract samples.

Four DNA probes were derived that hybridized specifically to DNA from Lactobacillus acidophilus O. The probes were constructed by randomly cloning lactobacillus DNA in plasmid vector pBR322. Two of the probes (pSR1 and pSR2) were composed of vector and plasmid DNA inserts (3.6 and 1.6 kb, respectively); the others (pSR3 and pSR4) were composed of vector and chromosomally derived inserts (6.9 and 1.4 kb, respectively). The probes were used to enumerate, by colony hybridization, strain O in digestive tract samples collected from piglets inoculated 24 hours previously with a culture of the strain. The probes did not hybridize to DNA from lactobacilli inhabiting the digestive tract of uninoculated piglets. Strain O made up about 10% of the total lactobacillus population of the pars esophagea and about 20% of the population in other digestive tract samples.     

Rapid preparation of vector-free hybridization probes suitable for screening recombinant libraries

A procedure is described to rapidly prepare radioactively labeled DNA inserts from crude recombinant plasmid DNA preparations. These probes can subsequently be used to identify homologous nucleotide sequences in bacteria containing recombinant plasmids by colony hybridization. In a single procedure, crude recombinant plasmid DNA is both ³²P-labeled and fragmented by nick-translation in the presence of sufficient pancreatic DNase I to produce radioactive DNA of about 0.2–0.3-kb single-strand length. At this DNA fragment length the majority of the vector and insert sequences are on different DNA fragments. The insert DNA can then be separated from vector and contaminating Escherichia colt host chromosomal DNA by the following method. The DNA fragment population is first denatured and renatured under conditions such that the recombinant plasmid DNA reassociates but host DNA does not. The renatured plasmid DNA fragments are separated from the denatured host DNA by hydroxylapatite chromatography. The plasmid DNA fragments are then denatured and renatured with an excess of insert-free vector DNA. Conditions are chosen such that the insert DNA remains single-stranded while the vector DNA becomes double-stranded. The single-stranded insert DNA can be separated from the double-stranded vector DNA on hydroxylapatite and used directly for colony hybridization.     

"In vitro" method of assembling a synthetic gene

Without prior in vitro enzymatic ligation a DNA duplex was assembled successfully by directly transforming competent cells with a mixture containing six synthetic complimentary oligodeoxyribonucleotides and a linearized plasmid. One out of 100 transformants was positive in colony hybridization with one of the synthetic fragment probe. The sequence of the DNA duplex inserted into the plasmid was confirmed by dideoxy sequencing method.     

The segregation of the 2 mu-based yeast plasmid pJDB248 breaks down under conditions of slow, glucose-limited growth

The stability of the 2 mu-based yeast plasmid pJDB248 in Saccharomyces cerevisiae S150-2B(cir0) was investigated in glucose-limited chemostat culture. Plasmid-free cells were detected by loss of (plasmid-encoded) leucine prototrophy and confirmed by colony hybridization. The plasmid was considerably more stable at a high dilution rate (0.12 h-1) than at a lower dilution rate (0.05 h-1). The average plasmid copy number in the cells retaining the plasmid remained constant at approximately 50 in the high dilution rate culture whereas it rose to almost 600 in the slow dilution rate culture. However, in both cultures the overall plasmid level in the total population remained constant, indicating that plasmid segregation breaks down at the low growth rate. Similar experiments on the native 2 mu plasmid demonstrated high stability and no significant differences between the high and low growth rate cultures. It is postulated that the difference in behaviour between the native and chimeric plasmids is related to an interaction between the growth conditions and the loss of the D gene product.     

A synthetic oligonucleotide probe and a cloned polynucleotide probe based on the yopA gene for detection and enumeration of virulent Yersinia enterocolitica

We compared a synthetically produced 19-mer oligonucleotide probe with a polynucleotide probe consisting of a cloned fragment of the virulence gene yopA for their relative efficiencies in identification and enumeration of virulent Yersinia enterocolitica. The probes were used in DNA-DNA colony hybridization assays to differentiate 70 Yersinia strains with known plasmid profiles. All 19 strains harboring the 40- to 50-megadalton virulence plasmid were positive in the hybridization assay, whereas their isogenic derivatives lacking this plasmid were negative. Both probes correctly identified plasmid-bearing variants of Y. enterocolitica serogroups O:3, O:5,27, O:8, O:9, O:13, and O:21 from three continents. In contrast, none of the probes hybridized with DNA from 32 environmental yersiniae belonging to 26 serogroups not associated with disease. Colony hybridization was used to detect and enumerate virulent Y. enterocolitica in three artificially contaminated food samples. Despite a large background of indigenous bacteria (3 x 10(4) CFU), the efficiency of enumeration ranged from 33 to 82%. The use of nylon filters did not impair the growth of virulent yersiniae. Both probes showed a perfect concordance in their specific differentiation and enumeration of virulent Y. enterocolitica. DNA colony hybridization with these two probes permitted rapid and reliable identification of all common pathogenic serogroups without the need for enrichment or esoteric identification protocols.     

A synthetic oligonucleotide probe and a cloned polynucleotide probe based on the yopA gene for detection and enumeration of virulent Yersinia enterocolitica.

We compared a synthetically produced 19-mer oligonucleotide probe with a polynucleotide probe consisting of a cloned fragment of the virulence gene yopA for their relative efficiencies in identification and enumeration of virulent Yersinia enterocolitica. The probes were used in DNA-DNA colony hybridization assays to differentiate 70 Yersinia strains with known plasmid profiles. All 19 strains harboring the 40- to 50-megadalton virulence plasmid were positive in the hybridization assay, whereas their isogenic derivatives lacking this plasmid were negative. Both probes correctly identified plasmid-bearing variants of Y. enterocolitica serogroups O:3, O:5,27, O:8, O:9, O:13, and O:21 from three continents. In contrast, none of the probes hybridized with DNA from 32 environmental yersiniae belonging to 26 serogroups not associated with disease. Colony hybridization was used to detect and enumerate virulent Y. enterocolitica in three artificially contaminated food samples. Despite a large background of indigenous bacteria (3 x 10(4) CFU), the efficiency of enumeration ranged from 33 to 82%. The use of nylon filters did not impair the growth of virulent yersiniae. Both probes showed a perfect concordance in their specific differentiation and enumeration of virulent Y. enterocolitica. DNA colony hybridization with these two probes permitted rapid and reliable identification of all common pathogenic serogroups without the need for enrichment or esoteric identification protocols.     

Selective enrichment of cDNAs from salt-stress-induced genes in the wheatgrass, Lophopyrum elongatum, by the formamide-phenol emulsion reassociation technique

We present a novel technique for the enrichment of cDNA libraries to enhance the abundance of clones of differentially expressed genes. The technique is relatively simple, requires moderate quantities of poly(A) + RNA and results in preferential enrichment of clones derived from mRNAs that were of low abundance in their original population. This method was used to isolate cDNA clones of salt-stress-induced genes in the roots of Lophopyrum elongatum, a highly salt-tolerant wheatgrass. An excess of sonicated plasmid DNA from a cDNA library from nonstressed roots was hybridized in a formamide-phenol emulsion with inserts from a cDNA library of stressed roots. Clones that were more abundant in, or were unique to, the library of the stressed roots were recovered as double-stranded fragments by virtue of reconstituted restriction-enzyme-digested ends by ligating them to a plasmid vector. The resulting enriched library was screened by differential colony hybridization and clones of eleven different genes that were more strongly expressed in stressed roots than in controls were selected.     

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. Send offprint requests to: A. Kandel at Department of Microbiology, Water Quality Institute, Agern Alle 11, DK-2970 Hørsholm, Denmark.     

Detection and enumeration of virulent Yersinia enterocolitica in food by DNA colony hybridization

A portion of a 44-megadalton plasmid found in Yersinia enterocolitica 8081 was used as a genetic probe to differentiate virulent and nonvirulent strains of the species. A DNA colony hybridization technique was employed. Three BamHI restriction endonuclease fragments labeled with 32P by nick translation were hybridized to lysed colonies of pure cultures, mixtures of virulent and nonvirulent cells, and portions of a food sample artificially contaminated with virulent Y. enterocolitica. The results of the colony hybridization test for virulence were the same as those obtained by the autoagglutination and suckling mouse tests. DNA colony hybridization detects pathogenic Y. enterocolitica in foods without the need for enrichment or pure cultures.     

Detection and enumeration of virulent Yersinia enterocolitica in food by DNA colony hybridization.

A portion of a 44-megadalton plasmid found in Yersinia enterocolitica 8081 was used as a genetic probe to differentiate virulent and nonvirulent strains of the species. A DNA colony hybridization technique was employed. Three BamHI restriction endonuclease fragments labeled with 32P by nick translation were hybridized to lysed colonies of pure cultures, mixtures of virulent and nonvirulent cells, and portions of a food sample artificially contaminated with virulent Y. enterocolitica. The results of the colony hybridization test for virulence were the same as those obtained by the autoagglutination and suckling mouse tests. DNA colony hybridization detects pathogenic Y. enterocolitica in foods without the need for enrichment or pure cultures.     

Genetic determinants of antibiotic resistance in enterobacteria

Antibiotic resistance of enterobacterial strains from population isolated in Krasnodar region is rather often controlled by the "plasmid" genes. The conclusion is based on using the colony hybridization with [32P]-DNA fragments of plasmids, carrying the genetic determinants of antibiotic resistance, as a method for antibiotic resistance, genes screening. Kanamycin resistance in the majority of strains is coded by APH (3') II gene, streptomycin resistance by APH (3") gene, chloramphenicol resistance by CATI, sulphonilamide resistance by DHPS type II gene. Tetracycline resistance of the studied enterobacterial strains is not connected with the widespread genetic determinants of a new class tetracycline resistance.     

Genetic study of plasmid integration into yeast chromosomes. V. Mapping of integration sites for the plasmid pYF91

The data on mapping the episomal plasmid integration sites in yeast chromosomes I, III, IV, V, VII, XV are presented. In addition to the integration site at leu2 of chromosome III localized earlier, 6 more loci containing apparently the homologous yeast transposons, with a copy in a plasmid, were defined. The fact of plasmid integration was proved by colony hybridization technique with the pBR322 probe. The plasmid DNA segregation (the ratio 2:2) and its linkage to pLEU2 plasmid marker gene were observed in hybrids of all integrants studied.     

Molecular cloning of the region of the terminus of Escherichia coli K-12 DNA replication.

A series of plasmids have been isolated either by ligation of defined restriction fragments to plasmid pBR325 or by screening of a cosmid bank by in situ colony hybridization. Together with one previously isolated plasmid, they spanned 86% of the 30.5- to 34-min region of the genetic map of Escherichia coli K-12. Physical analysis of these plasmids and hybridizations to Southern blots confirmed the endonuclease map of this region, with the exception of a 9.3-kilobase pair inversion.     

Screening of cloned recombinant DNA in bacteria by in situ colony hybridization.

We have developped in situ methods of colony hybridization in which there is no need to replicate colonies one by one prior to hybridization. The best method consists in promoting partial lysis of the colonies on the plates by means of a resident thermoinducible prophage. It appears that colonies are heterogeneous with respect to prophage induction, so that survivors remain in each colony. Blotting onto nitrocellulose filters and hybridization with a highly radioactive probe permits the screening of many thousands of colonies per plate for the presence of a DNA sequence carried by a plasmid and complementary to the probe. This procedure greatly facilitates the isolation of recombinant plasmids which carry a specific DNA sequence. We also describe a second, less efficient procedure which does not use prophage induced lysis, and is potentially usable with B2 or EK2 safety systems, without modification of the bacterial hosts.     

Cloning and expression of the yeast galactokinase gene in an Escherichia coli plasmid.

This report describes the construction and isolation of a plasmid, derived from pBR322, which carries a BglII restriction fragment of DNA containing the galactokinase gene from Saccharomyces cerevisiae. This was accomplished by the following procedure: (1) Purified galactokinase mRNA, labelled with 125I, was hybridized to BglII digests of yeast DNA employing Southern's filter transfer technique to identify a restriction fragment containing the galactokinase gene. (2) This fragment was partially purified by agarose gel electrophoresis, ligated into the BamHI site of pBR322 and transformed into Escherichia coli to generate a clone bank containing the galactokinase gene. (3) This bank was screened by in situ colony hybridization with galactokinase mRNA resulting in the identification of a plasmid carrying this gene. This plasmid DNA hybridized with the galactokinase mRNA to the same extent in the presence of absence of a large excess of unlabelled mRNA from cells that were not induced for galactokinase synthesis, while the same amount of unlabelled galactose-induced mRNA reduced the hybridization by 95%. When this plasmid was introduced into an E. coli strain deleted for the galactose operon it caused the synthesis of low levels of yeast galactokinase activity.     

Molecular cloning of human immune interferon cDNA and its expression in eukaryotic cells.

Starting with mRNA derived from Staphylococcal enterotoxin A induced human splenocytes, dsDNA was synthesized and inserted into unique BamHI site of the eukaryotic expression vector pSV529 (1). A recombinant plasmid containing human immune interferon (IFN-gamma) cDNA was identified by hybridization of plasmid inserted DNA bound onto nitrocellulose filters with mRNA derived from SEA-induced splenocytes, translation of the eluted RNA in Xenopus laevis oocytes and assaying for IFN activity. Plasmids containing the entire human IFN-gamma cDNA sequence were identified by colony hybridization and were sequenced. A unique coding region was identified which predicted a protein of 166 amino acids, the 20 N-terminal amino acids of which presumably represent a signal peptide. After transfection of monkey cells with plasmid DNA isolated from one of the recombinant clones (pHIIF-SV-gamma 1), IFN was excreted into the culture medium. This IFN was not distinguishable from human IFN-gamma by serological criteria or by cell target species specificity.