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...

Conclusion

Antibiotic resistance genes have a long history of reliability for use as highly selective, versatile markers allowing detection and enumeration of bacteria in environmental samples. Resistance genes found in non-antibiotic producing bacteria are readily expressed in a wide range of host backgrounds. Examples include the neomycin resistance gene, nptII, which has been used in gram-positive (high and low GC) and gram-negative bacteria, yeasts, plant and animal cell cultures. The resistance genes...

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

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...

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...