Luc Based Systems

Although bacterial luciferases are most fEquently used as markers for environmental use, strains carrying the firefly luciferase or other insect luciferase genes can provide advantages. The enzymes from insects are in general more heat stable, are not subject to substrate inhibition and their quantum yield (90 ) is considerably higher than that of bacterial luciferase (5-10 ). Firefly luciferase requires ludferin, a heterocyclic carboxylic acid, oxygen and ATP as substrates. The requirement for...

Conclusion

The GUS system is a precise and robust reporter for gene expression, allowing transcriptional and translational fusions between the gene of interest and the gusA gene. The main advantages of GUS are the absence of endogenous activity in plants, the existence of a large number of commercially available substrates and the number of constructs facilitating the use of the E. coli gusA gene. GUS has been used both as a reporter and marker in studies with rhizosphe*e bacteria, e.g., the...

Detection of RNA

Analysis of Gene Expression on the Basis of mRNA Assessments Analysis of mRNA directly extracted from environmental samples is conducted to investigate habitat dependent regulations of gene expression. In eukaryotic organisms, transcribed mRNA molecules are altered by various co- and posttranscriptional modifications such as capping of the 5' end and polyadenylation of the 3'-end.90 These modifications increase the stability of the mdecules to half-life values of several hours. The...

Effect of Plasmids on Entry into the VBNC State

To make the situation potentially worse, it now appears that our knowledge of what induces the VBNC state in various bacteria must be re-examined, taking into account whether or not the cells under investigation harbor extrachromosomal plasmids. We have reported10 that Pseudomonas fluorescens cells enter the VBNC state when incubated in sterile river water at 37 C, but not at 5 or 25 C (Fig. 1.8A). In rather remarkable contrast, when this same strain harbored a plasmid (pFAC510), the cells...

Eva Tas and Kristina Lindstrom 41 Introduction

The detection and identification of specific bacterial species and strains has long been of interest for different applications in agriculture, the food industry, the bioleaching industry and the biological degradation of toxic wastes (bioremediation). In addition, basic and applied sciences, such as microbial ecology, are investing more research into exploring the components of microbial populations living in various ecological niches. Classical microbial identification methods are primarily...

Extraction of Microbial Cells from Environmental Matrices

Recovery and concentration of microbial cells may be an important first step if nucleic acid analysis is to be performed in environments with relatively low cell densities, such as aquatic or phyllosphere habitats. Moreover, even for environments with high population densities, it can be advantageous to prepare a cell fraction prior to nucleic acid extraction, in particular if intracellular targets are to be assessed. Bacterial diversity assessments based on DNA reassociation kinetics21 even...

Field Release

The release site (Fig. 11.2), a 3 x 3 m plot within the site of the RSM2004 release6 had been under rotation with cereal crops since 1989. To comply with the requirements of the U.K. Department of the Environment, the plot was covered by a net to exclude birds and surrounded by two 1 m buffer zones of wheat separated by a fence. The release took place in July 1994 inoculant granules were placed in drills (10 cm apart), into which 840 untreated Avola peas (PGRO, Peterbourgh, U.K) coated in...

Expression Vectors Using Eukaryotic Luciferases as Bacterial Markers

The eukaryotic nature and presumed absence of firefly and click beetle luciferase genes may provide a unique genotype to bacteria and the variation in wavelength of emitted light provides an additional advantage. To exploit this difference in luciferase assays, luc and lucOR genes, which encode emission of light at 560 nm (yellow-green) and 595 nm (orange), respectively, have been used to develop marker genes for bacteria employing three transcriptional units (Fig. 5.3). The first, an...

Introduction

Molecular marker systems provide the ability to track specific microbial inocula in the environment. This is a fundamental requirement for many ecological studies and is particularly appropriate for the assessment of risks associated with the release of genetically modified microorganisms. The efficiency of a marker system requires the absence of the marker gene, or its phenotype, from the environment under study, stable insertion of the marker genes into the host oganism without loss cf...

Heavy Metal Sensors

Regulatory elements from bacteria resistant to a heavy metal can provide a sensitive and selective receptor for heavy metal bioavailability53, 54 which will function in physiological concentration ranges. Genetic determinants for heavy metal resistance are usually found on plasmids and transposons of soil bacteria, which facilitate their analysis and manipulation by molecular genetic techniques. Many have been characterized, including mercury,55 arsenite,56 cadmium,57,58 zinc,59 cobalt,59...

Root Colonization and Systemic Spreading of Azoarcus sp Strain BH72

Azoarcus sp.42 is an endorhizospheric isolate of Kallar grass. Kallar grass is an undo-mesticated C4 plant with high tolerance to soil salinity, alkalinity and waterstress. Similarly as has been described for Azospirillum, colonization of Kallar grass by Azoarcus has been studied using a constitutively expressed gusA fusion. For this, mutants of Azoarcus were generated by transposon mutagenesis with Tn5-gusA1 (Table 6.1) and selected for constitutive GUS expression.33 Microscopic examination of...

Supply of Aldehyde for the Bioluminescent Reaction

The bioluminescent reaction requires a long chain aldehyde substrate and tetradecanoic acid appears to be the precursor of the natural substrate tetradecanal.10 A fatty acid reductase complex, responsible for aldehyde synthesis, consists of two distinct functional components 11 acyl protein synthase (50 kDa) which, with cleavage of ATP to AMP, activates the fatty acid to a fatty acyl intermediate, and acyl-protein reductase (58 kDa), responsible for the transfer of the acyl group from the...

Analysis of DNA and Detection of Specific Sequences

Commony, the quantity and quality of extracted environmental DNA is analyzed by standard methods, such as absorbance at 260 nm in the spectrophotometer or gel electrophoresis.1,2,54 However, due to the presence of coabsorbing compounds (humic adds) in most nucleic acid extracts,14,15,20 direct spectophotometry is not recommended. A solution is offered by DNA-intercalating compounds such as Hoechst dye 33258, which results in DNA absorbance at different wavelengths.2,11,20 Gel electrophoresis,...

Brief Description of PCRBased Fingerprinting Methods

PCR based fingerprinting methods are useful for bacterial identification and, as such, are gaining an important role in ecological studies. Some cf these methods, such as arbitrarily primed PCR8 and random primed amplified polymorphic DNA (RAPD) analysis,9 are based on amplification of multiple DNA fragments from genomic DNA with random or arbitrary primers, which will result in a characteristic pattern of PCR products for each different organism. Amplified fragment length polymorphism (AFLP)...

Concern on the Use of Hazardous Chemicals in Connection with the Deliberate Release of a GMM

The use of hazardous chemicals in relation to GMMs raises concern. Concerns and risk assessments of GMOs tend to be quite broad, which must be due to the accumulated knowledge on risks from other areas. GMMs must often be used in connection with chemicals, e.g., as above due to selectable marker genes or for post-release cleanup. Methyl bromide has been suggested for post-release clean up. The use of very hazardous chemicals should in general be avoided in post-release treatment after field...

Conclusions and Perspectives for Further Development

From a practical perspective, several of the DNA and RNA extraction and analysis methods discussed herein are suitable for the screening of large sample numbers. They are thus useful for the assessment of the fate of GMMs and or thar genes (activity) following introduction into environmental matrices. If required, they also facilitate the detection of naturally occurring DNA or RNA sequences in soil microbes. Due to the cuirent lack of comprehensive data with respect to the suitability of...

Cultivation Based Assays

The traditional approach for monitoring bacteria using antibiotic resistance is selective agar plate counting, although the most probable number technique (MPN) can be used for selective broth culture. Agar antibiotic gradient plates, in which the antibiotic concentration increases linearly from zero to an established level, are produced by making an agar gradient containing the antibiotic and covering a basal agar layer without antibiotic. Such gradients are difficult to replicate and have not...

Detection of Strains CT0370 and RSM2004

Routine CT0370 culturing was on complete TY or minimal Y medium, detailed by Hirsch and Skinner,14 at 28 C. To count CT0370 in soil, selective TY or Y agar were used, containing 500 g ml-1 streptomycin (Str) and 200 g ml-1 spectinomycin (Sp) to counterselect other bacteria, cycloheximide 100 g ml-1 and benomyl at 7.5 g ml-1 to inhibit fungi, and the GUS substrate X-gluc cyclohexamine salt, supplied by NBL Biologicals, U.K.) at 50 g ml-1. In soil from the release site before inoculation there...

Extraction and Analysis of Microbial Community Nucleic Acids from Environmental Matrices

Jan Dirk van Elsas, Kornelia Smalla, Christoph C. Tebbe 3.1. Introduction Environmental monitoring on the basis of nucleic acids is increasingly being recognized as an extremely powerful approach, since organisms or genes can be directly assessed, even without prior cultivation. Hence, targets present in unculturable, poorly-culturable or as yet uncultured organisms, which would escape detection when using traditional cultivation-based approaches, are assessable. In the last decade, a number of...

Ethical Concerns

Although there are obvious advantages in the use of antibiotic resistance gene markers, their value for field releases has been limited. This is probably due to the ethical questions raised about deliberately releasing resistance genes into the environment, which presents the risk of spread into the indigenous bacterial population. However, transfer is known to be limited in conditions of low nutrients24,16 and is also affected by the activity and spatial distribution of bacteria in addition to...

Experimental Design What Can Be Achieved with Each System

Three different successive experimental setups were used at the FAL (Braunschweig) to study the survival and ecological fitness of tagged strains, S. meliloti L1 and L33 (Fig. 9.2). The first experimental setup consisted of soil columns in the greenhouse, the second were field lysimeters of the same size as the greenhouse soil columns and the third setup were field plots which were inoculated with the respective S. meliloti strains. Characteristics of the three setups are shown in Table 9.1....

Extraction and Purification of RNA

In contrast to DNA, which serves as a pool of genetic information, RNA has multiple cellular functions. Generally, three classes of RNA can be distinguished, i.e., messenger (m)RNA, ribosomal (r)RNA and transfer (t)RNA. mRNA molecules are transcribed from genes during gene expression. These transcripts are translated into amino add sequences at ribosomes, which are made up cf rRNA molecules and p-oteins. tRNA molecules serve as carriers for amino adds in this translation process in protein...

Field Trial I Fate and Impact of an Allochtonous GMM

Upon authorization from the national competent authority, the three GM rhizobia tested in microcosms were released as field inoculants in the presence of their pea host plant (Pisum sativum) by liquid inoculation at about 5x106 cfu per seed. The release took place in soil at the agricultural experimental station of the University of Padova.5 Bacteria were monitored in soil, rhizosphere and nodules. The parental strain 1003, from which the GMMs were derived had been isolated in northern Europe...

Future Prospects

Nucleic acid based methods for assessment of the environmental fate and effects of bacteria are under continuous development. In the future, growth and wider availability of nucleic acid sequence databases, further improvement and optimization of molecular techniques as well as application of microarray chips will facilitate monitoring studies. DNA databases are of great help in probe and primer design. Since data are accumulating very fast in nucleic acid sequence databanks, special databases...

GFP as a Bioreporter in Bacteria

The fusion of GFP to a protein of interest and monitoring of the expression cf the fusion product in vivo permits the assessment of the cellular localization of the fusion protein (For reviews see refs. 3,5,6,8). Although GFP looses its fluorescence when truncated,3 the full-length protein can be used successfully in both C-terminal and N-terminal protein fusions54 GFP has been used extensively as a bioreporter to study cell division in bacteria. For example, Ma et al have created fusions of...

GUS Constructs of Wilson et al25

Wilson et al25 constructed vectors useful for manipulation of gusA and a whole series of minitransposons containing gusA as a reporter gene. The vectors allow construction of translational fusions to gusA in all three open reading frames. In the miniTn5, constructed by de Lorenzo et al26 and Herrero et al,27 transposase activity inherent to Tn5 has been deleted allowing a stable insertion. Transposase activity has, thus, to be provided in trans for transposition. Wilson et al25 used the unique...

Inducing Factors

Different bacteria are known to enter the VBNC state in response to different factors, all of which a cell would normally encounter in natural environments. These include such stresses as elevated or reduced temperature, elevated or reduced osmotic (e.g., salt) concentrations, nutrient starvation, levels of oxygen, and even certain intensities of light.1 In all cases, the inducers of the VBNC state appear to be environmental factors which are potentially injurious to a given bacterial species....

Photon Imaging

Quantitative imaging of gene expression using lutferase based reporter constructs is now possible due to the availability of extremely sensitive imaging equipment that can be calibrated using light standards. These camera systems are usually based on image intensifi-ers coupled to video came*as, or cooled charge coupled device (CCD) technology. Intensified cameras have high sensitivity and the ability to watch the image form almost in realtime but, at high gain, suffer from reduction in spatial...

Plant Growth Promoting Bacteria PGPR of the Genus Azospirillum

Bacteria living in the rhizosphere and favorably affecting plant growth are denominated Plant Growth Promoting Rhizobacteria (PGPR). PGPR of the genus Azospirillum were initially studied because of their association with Gramineae and because of their ability to fix atmospheic nitrogen.38 Field trials, however, indicated that bacterial nitrogen is not responsible for the enhanced plant growth observed upon Azospirillum inoculation.39 The observation that inoculation of plant roots with...

Preface

Environmental microbiology is currently one of the most rapidly expanding areas of scientific research. Impetus for advanced investigations has been provided by the development and application of molecular techniques that facilitate the identification, characterization and monitoring of microbes. These advances now allow detailed investigations, developed in the laboratory, to be undertaken in the natural environment. Such studies confirm the remarkable biological diversity represented by...

Properties of Wildtype GFP

Wild-type GFP (wt GFP) is a 27 kDa monome consisting of 278 amino acid residues.1 In Aequorea victoria, GFP serves to convert blue chemiluminescence, generated by the protein aequorin, into green light.12 This conversion is accomplished by means of an internal protein p-hydroxybenzylideneimidazolinone chromophore formed by the cyclization of three amino acid residues of GFP, Ser-Tyr-Gly, followed by oxidation of the Tyr residue. These essential three residues are located at positions 65-67 cf...

Purification

The aim of purification of the crude extract is to recover the nucleic acids from the crude extract in a form ready for analyses by appropriate molecular techniques. Ideally, the procedure is as rapid and simple as possible, and incurs minimal quantitative and or qualitative loss of, or damage to, the nucleic acids. The different protocols currently in use in different laboratories have been based on an array of different steps in various combinations. Table 3.1 presents a summary of...

References

Identification of genes and gene products necessary for bacterial bioluminescence. Proc Natl Acad Sci USA 1984 81 4154-4158. 2. Eberhard A, Hastings JW. A postulated mechanism for the bioluminescent oxidation of reduced flavin mononucleotide. Biochem Biophys Res Comm 1972 47 348-353. 3. Kurfurst M, Ghisla S, Hastings, JW. Characterization and postulated structure of the primary emitter in the bacterial luciferase reaction. Proc Natl Acad Sci USA 1984 81 2990-2994. 4....

Quantification of Specific Targets in Environmental Nucleic Acids

Specific targets in environmental DNA or RNA can be directly quantified by hybridization in a dot blot setup, using careful calibration with a dilution range of the target in the same background.64 Among numerous other applications, the fate in soil of a genetically marked self-transmissible plasmid, RP4, has thus been successfully monitored.56 Quantification of specific mRNA can also be achieved by hybridization with a single-stranded RNA probe. To assess the level of gene expression, the...

Rhizobiumlegume Symbiosis

Sharma and Signer23 have used Tn5-gusAi and Tn5-gusA2 to study the expression of Rhizobium meliloti symbiotic genes during nodule organogenesis of alfalfa. Two patterns of spatial and temporal expression of the symbiotic genes were observed. The nod genes, involved in nodule formation, are expressed in an early phase (from 36 hcurs after inoculation) on the root surface and nodule cortex and later (from 10 days after inoculation) especially at the nodule meristem. In contrast, the nif (encoding...

Screening for pSym Acquisition by CT0370 in the Field

Pea plants were harvested at pod-fill, the roots washed in water, nodules removed and surface sterilized by washing in 70 ethanol for 30 s, rinsing in H2O, leaving in 10 sodium hypochlorite for 1 min, then washing twice in H2O. They were stored at -70 C in sterile 96 well microtiter dishes containing sterile 10 glycerol. The nodules were screened for GUS activity using MUG (4-methyl-umbelliferyl-p-D-glucoronide supplied by NBL biologicals) and UV-induced fluorescence was assessed as...

Significance of the VBNC State in the Release of Genetically Modified Bacteria

There are numerous practical consequences of the existence of the VBNC state in bacteria. One example is the fact that coliforms, the assay for which is employed worldwide as an indicator of fecal contamination of drinking and recreational waters, also enter the VBNC state. Entrance of these bacteria into this dormant state appears to result from exposure to low temperatures or to increased salt levels. Thus, it must frequently be the case that waters are considered to be coliform negative...

Specificity of Selected Probes and Primers

Specificity of each probe and primer pair has to be experimentally evaluated, especially against closely related nontaiget groups of bacteria or bacteria inhabiting the environment studied. Since the probes are based on the sequences of strains from culture collections, we cannot be sure that they recognize all possible organisms from an environmental sample that belong to a target group. For example, the developed probes or primers might not detect organisms of environmental importance, which...

Survival of GMM on Over Wintering Sugar beet Secondary Growth Resown Crop Plants and Volunteer Weeds

No GMMs were detected in samples of over-wintered leaf rosettes and the rhizospheres of the sugar beet plants collected in mid March 1994 (11 months after sowing). When samples of new secondary leaf and rhizosphere growth were collected 384 days after sowing of the inoculated seeds, GMMs were detected (Table 8.2). These old plants were removed to prevent bolting and new sugar beet seed was sown in the recombinant treated plots. The new seedlings and subsequent plants were assessed for the...

Techniques and Consequences of GMM Soil Inoculations

In all three experimental setups it was intended to start the experiment with approx. 106 cells of the strain g-1 soil in the upper soil horizon (plough layer 0 to 25 cm depth). In greenhouse experiments, batch culture grown cells were harvested, washed in potassium phosphate buffer (50 mM, pH 7.2), and mixed with sterile peat and soil taken from the upper 4 cm of the soil columns. This mixture was loaded onto the soil columns and alfalfa seeds were added. A similar technique was applied to...

The Tn5gusA Promoter probe transposons of Sharma and Signer23

Sharma and Signer23 constructed Tn5-gusA promoter probe transposons to monitor expression cf Rhizobium meliloti symbiotic genes within alfalfa nodules. For these transposons, a Tn5 derivative,24 in which 25 base pairs of IS50L are retained, was used. Tn5-gusA1 is a transcriptional operon fusion tansposon. The gusA gene is preceded by translation stop codons in al open reading frames and a Shine-Dalgarno sequence to allow initiation of gusA translation. Tn5-gusA2 is similar although one of the...

The Viable but Nonculturable State

Microbial ecologists have long recognized that large proportions of microbial populations inhabiting natural habitats appear to be nonculturable. Indeed, plate counts of bacteria in soil, rivers and oceans typically indicate that far less than 1 of the total bacteria observed by direct microscopic examination can be grown on culture media. It has also long been known that certain portions of bacterial populations in natural environments seem to disappear during certain seasons, only to reappear...

United States

The responsible agency for regulatory developments in biotechnology in the US is the United States Environment Protection Agency (EPA) concerning microbial plant pesticides, new uses of existing pesticides and novel microorganisms.g The relevant laws are the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) and the Toxic Substances Control Act (TSCA). In 1986, the US Office of Science and Technology Policy published a co-ordinated framework for regulation of biotechnology in the...

Use of Randomly Cloned Fragments as Specific Probes

When designing hybridization probes, it is not essential to know the function of the selected DNA fragment(s). Randomly cloned DNA fragments, such as entire or enriched genomic libraries, or sub-fragments from partially described genomic regions can be screened for unique sequences.41 Alternatively, specific sequences can be isolated by differential comparison of two or more genomes . Subtraction hybridization, which will be discussed later in more detail, is one method based on this idea....

GFP as a Biomarker in Bacteria

GFP has been used as a biomarker in a variety of bacterial strains. A partial list of the bacterial species studied and the types of studies performed is presented in Table 7.2. Use of constitutive or inducible promoters to express GFP in bacterial cells allows tracking of tagged cells in the environment and visualization of their location.10 Constitutive expression can be obtained by cloning GFP into a vector containing a promoter known to be oonstitutively expressed in the bacterium of...

Case Study of the Persistence of P fluorescens SBW25EeZY6KX in Sugar beet Crops

Data gathered in microcosm and field investigations confirmed the utility of P. fluorescens SBW25EeZY6KX as a competitive phytosphere colonizer of plants when introduced as a seed dressing4,5,19 or foliar spray.20,21 In open field studies, information on the survival, establishment and dissemination of GMMs in the environment was acquired during the development and maturation of deliberately inoculated field crops. Data on the spread, persistence and survival of the inocula on plants and in...

The GUS Reporter system

Going back to the landmark publication of Jefferson et al,15 we can only now put in perspective to what extent the use of the Escherichia coli gusA gene as a reporter shifted the balance from reporter systems suited only for specific studies to a broad purpose, easy-to-use, and precise reporter of gene expression. The use of fusions between a gene of interest and a reporter gene with an easily detectable phenotype, such as gusA, offers several advantages for the study of gene expression. Two...

Improving GFP Fluorescence

GFP from the sea pansy Renilla reniformis contains the same chromophore as GFP from Aequorea victoria. However, the GFP from R. reniformis has a single excitation peak at 498 nm. This difference is presumably due to the difference in amino acid residues surrounding the (hromophore in the mature protein.15 Neither the isolated chromophore, nor the denatured protein at neutral pH, are fluorescent.13 These data suggested that changing the protein environment (amino acid sequence) around the...

Marking of Bacteria with Antibiotic Resistance Genes

Chromosomes cf bacteria are often marked by the use of random or site-specific integration vectors, such as transposons. Location of marker genes on the chromosome instead of plasmids may improve stability, and although this reduces the likelihood of a transfer event, it does not eliminate it. Insertion of markers into the chromosome has been achieved by random integration of transposons which encode resistance genes, such as Tn50i in E. coli,33 Tn903 in Erwinia carotovora,34 Tn5 in...

Cell Disruption

The aim of the cell disruption step is to lyse as many target microbial cells as possible, since any subsequent analysis on the basis of nucleic acids is greatly sensitized if this step is optimized. The lysis protocol can be targeted towards a specific microbial group or to the total microbial community. Unfortunately, cell lysis is often the main limiting step in nucleic acid extraction protocols, even of pure cultures.46 Due to the enormous differences in lysability of cells (cf. easily...

The Use of Antibiotic Resistance Gene Markers for Studying Bacterial Populations in Natural Environments

Sharon Egan and Elizabeth M H Wellington 2.1. Introduction Antibiotic resistance genes have been used to mark bacteria by providing a readily selectable phenotype, which can be detected using selective growth media. Detection and monitoring is therefore culture-dependent. A wide range of resistance genes have been characterised (Tables 2.1 and 2.2) which confer resistance to commercially available, inexpensive antibiotics. In addition, resistance genes have provided a valuable tool for cloning...

Choice of Antibiotic Resistance Gene Markers

The choice of resistance gene markers depends on the environmental use of the marked bacterium and is influenced by the background resistance of the indigenous bacteria in a given habitat. Members of some genera, including Pseudomonas, Mycobacterium and Rhodococcus, have nonspecific mechanisms for resistance to antibiotics and heavy metals, including exclusion from the cell.16 The levels of resistant indigenous bacteria depends on the antibiotic and can range from 103-104 cfu g-1 dry soil for...

Use of Antibiotic Resistance Genes to Monitor Gene Transfer in Soil

Antibiotic resistance markers have been used to study gene mobility and dissemination of antibiotic resistance genes has been the subject of a large number of studies due to the problem of drug resistance in bacterial pathogens. Resistance genes are highly mobile under intense selection pressure in clinical environments and have been found associated with chromosomes, plasmids, integrons and bacteriophages.38,39 The application of antibiotics such as streptomycin to treat bacterial rots in soft...

The Escherichia coli gusA Gene

Coli Internal

Originally, p-glucuronidase was biochemically characterized in the bacterium Escherichia coli14 and later the gusA gene was isolated from E. coli strain K12.15 GUS activity enables E. coli in its natural environment, the gut, to decouple glucuronic acid from various substrates13 and to use glucuronic acid for further metabolization. The compounds coupled to glucuronic acid, the aglycones, can be very diverse. They are coupled to glucuronic acid in the liver to make them more water-soluble and...

Characteristics of Cells in the VBNC State

Cells entering the VBNC state generally undergo a reduction in size. In the case of V. vulnificus, for example, whereas log phase actively growing cells might be 3 mm long, those in the VBNC state are typically 0.6 m in diameter. During this size reduction, significant changes in membrane structure, protein composition, ribosomal content, and possibly even DNA arrangement are experienced. Again using V. vulnificus as an example,1 we have found rapid and dramatic decreases in the levels of...

Other GUS Constructs

Five gusA cassettes, uidAl, uidA2, uidA2-cat, uidA2-aadA and uidA2-aph, suitable for constructing transcriptional fusions,were described by Metcalf and Wanner.28 Three of them contain additional antibiotic resistance genes see Table 6.3 . uidAl, uidA2 and uidA2-aph cause nonpolar mutations after double homologous recombination into the host genome. Table 6.3. Overview of GUS constructs used in studies of plant-bacteria interactions gusA-expressing transposon gusA-expressing transposon Promoter...

Resuscitation from the VBNC State

For the VBNC response to represent a true survival response it must be possible for the cells to exit this dormant state and return to a fully active and culturable state. Such a reversal in physiology is termed resuscitation, and often is triggered simply by the removal of the stress which initially induced the VBNC response. In V. vulnificus, for example, exit from the low temperature-induced VBNC state is triggered by a temperature upshift e.g., from 5 C to room temperature . After such a...

Application of the R galegae Specific Probes and Primers for Monitoring

To study the applicability of detection with strain- and species-specific sequences, a greenhouse experiment was conducted.51 Soil devoid of R. galegae was inoculated with different levels of R. galegae HAMBI 1207, which forms an ineffective symbiosis with G. orientalis. Then seeds of G. orientalis, inoculated with the effective strain R. galegae HAMBI 1174, were sown into the soil. The competition between the two Rhizobium strains was assessed by measuring plant dry weights from the plots and...