RESUMO
BACKGROUND: Contamination from fecal bacteria in recreational waters is a major health concern since bacteria capable of causing human disease can be found in animal feces. The Dog Beach area of Ocean Beach in San Diego, California is a beach prone to closures due to high levels of fecal indicator bacteria (FIB). A potential source of these FIB could be the canine feces left behind by owners who do not clean up after their pets. We tested this hypothesis by screening the DNA isolated from canine feces for the bacteriophage-encoded stx gene normally found in the virulent strains of the fecal bacterium Escherichia coli. RESULTS: Twenty canine fecal samples were collected, processed for total and bacterial fraction DNA, and screened by PCR for the stx gene. The stx gene was detected in the total and bacterial fraction DNA of one fecal sample. Bacterial isolates were then cultivated from the stx-positive fecal sample. Eighty nine of these canine fecal bacterial isolates were screened by PCR for the stx gene. The stx gene was detected in five of these isolates. Sequencing and phylogenetic analyses of 16S rRNA gene PCR products from the canine fecal bacterial isolates indicated that they were Enterococcus and not E. coli. CONCLUSIONS: The bacteriophage-encoded stx gene was found in multiple species of bacteria cultivated from canine fecal samples gathered at the shoreline of the Dog Beach area of Ocean Beach in San Diego, California. The canine fecal bacteria carrying the stx gene were not the typical E. coli host and were instead identified through phylogenetic analyses as Enterococcus. This suggests a large degree of horizontal gene transfer of exotoxin genes in recreational waters.
RESUMO
Many bacteria produce secreted virulence factors called exotoxins. Exotoxins are often encoded by mobile genetic elements, including bacteriophage (phage). Phage can transfer genetic information to the bacteria they infect. When a phage transfers virulence genes to an avirulent bacterium, the bacterium can acquire the ability to cause disease. It is important to understand the role played by the phage that carry these genes in the evolution of pathogens. This is the first report of an environmental reservoir of a bacterial exotoxin gene in an atypical host. Screening bacterial isolates from the environment via PCR identified an isolate with a DNA sequence >95% identical to the Staphylococcus aureus enterotoxin A gene (sea). 16S DNA sequence comparisons and growth studies identified the environmental isolate as a psychrophilic Pseudomonas spp. The results indicate that the sea gene is present in an alternative bacterial host, providing the first evidence for an environmental pool of exotoxin genes in bacteria.
RESUMO
The ease of rapidly accumulating a large number of mutants requires careful bookkeeping to avoid confusing one mutant with another. Each mutant constructed should be assigned a strain number. Strain numbers usually consist of two to three capital letters designating the lab where they were constructed and a serial numbering of the strains in a central laboratory collection. Every mutation should be assigned a name that corresponds to a particular gene or phenotype, and an allele number that identifies each specific isolate. When available for a particular group of bacteria, genetic stock centers are the ultimate resources for gene names and allele numbers. Examples include the Salmonella Genetic Stock Centre ( http://www.ucalgary.ca/~kesander/), and the E. coli Genetic Stock Center (http://cgsc.biology.yale.edu/). It is also important to indicate how the strain was constructed, the parental (recipient) strain, and the source of any donor DNA transferred into the recipient strain (Maloy et al., 1996).
Assuntos
Genes Bacterianos , Terminologia como Assunto , Alelos , Cromossomos Bacterianos , Elementos de DNA Transponíveis , DNA Bacteriano , Mutação , PlasmídeosRESUMO
One of the greatest advances in molecular genetics has been the application of selectable transposons in molecular biology. After 30 years of use in microbial genetics studies, transposons remain indispensable tools for the generation of null alleles tagged with selectable markers, genetic mapping, manipulation of chromosomes, and generation of various fusion derivatives. The number and uses of transposons as molecular tools continues to expand into new fields such as genome sciences and molecular pathogenesis. This chapter outlines some of the many uses of transposons for molecular genetic analysis and strategies for their use.
Assuntos
Elementos de DNA Transponíveis , Mutagênese Insercional , Antibacterianos/farmacologia , DNA Bacteriano/análise , Resistência Microbiana a Medicamentos/genética , TransposasesRESUMO
Transposon insertions in and near a gene of interest facilitate the genetic characterization of a gene in vivo. This chapter is dedicated to describing the isolation of mini-Tn10 insertions in any desired nonessential gene in Salmonella enterica, as well as the isolation of mini-Tn10 insertions near particular genes. The protocols describe use of a tetracycline-resistant Tn10 derivative, but similar approaches can be used for derivatives resistant to other antibiotics. In addition, these approaches are directly applicable to other bacteria that have generalized transducing phages.
Assuntos
Elementos de DNA Transponíveis , Mutagênese Insercional , Genes Bacterianos , Salmonella enterica/genética , Transdução GenéticaRESUMO
Localized mutagenesis can be used to obtain mutants in genes of interest based on linkage to selectable markers. Mutagens diethylsulfate and hydroxylamine are used to obtain predominantly transition mutations in the DNA either by whole chromosomal mutagenesis or mutagenesis of DNA isolated as purified plasmid or packaged in transducing phage particles. Selectable markers can include those based on auxotrophic requirements, carbon or nitrogen source utilization, or antibiotic resistance markers, such as those encoded in transposons.
Assuntos
Mutagênese , Cromossomos Bacterianos , DNA Bacteriano , Genes Bacterianos , Mutagênicos , MutaçãoRESUMO
Coupling the expression of a gene with an easily assayable reporter gene provides a simple genetic trick for studying the regulation of gene expression. Two types of fusions between a gene and a reporter gene are possible. Operon fusions place the transcription of a reporter gene under the control of the promoter of a target gene, but the translation of the reporter gene and target gene are independent; gene fusions place the transcription and translation of a reporter gene under the control of a target gene, and result in a hybrid protein. Such fusions can be constructed in vitro using recombinant DNA techniques or in vivo using transposon derivatives. Many different transposon derivatives are available for constructing operon and gene fusions, but two extremely useful fusion vectors are (1) Mu derivatives that form operon and gene fusions to the lacZ gene, and (2) Tn5 derivative that forms gene fusions to the phoA gene.
Assuntos
Regulação Bacteriana da Expressão Gênica , Fusão Gênica , Óperon , Salmonella/genéticaRESUMO
The bacteriophage P22-based challenge system is a sophisticated genetic tool for the characterization of sequence-specific recognition of DNA and RNA in vivo. The construction of challenge phage follows simple phage lysate preparations and detection of constructs by positive selection methods for plaques on selective strains. The challenge phage system is a powerful tool for the characterization of protein-DNA and protein-RNA interactions in vivo. The challenge phage has been further developed to characterize the interactions of multiple proteins in heteromultimeric complexes that are required for DNA binding. Under appropriate conditions, expression of the ant gene determines the lysis-lysogeny decision of P22. This provides a positive selection for and against DNA binding: repression of ant can be selected by requiring growth of lysogens, and mutants that cannot repress ant can be selected by requiring lytic growth of the phage. Thus, placing ant gene expression under the control of a specific DNA-protein interaction provides very strong genetic selections for regulatory mutations in the DNA-binding protein and DNA-binding site that either increase or decrease the apparent strength of a DNA-protein interaction in vivo. Furthermore, the challenge phage contains a kanamycin-resistance element that can be used to either directly select for lysogeny or to determine the frequency of lysogeny for a given protein-DNA interaction to measure the efficiency of DNA binding in vivo. Selection for lysogeny can be used to isolate DNA-binding proteins with altered or enhanced DNA-binding specificities. The challenge phage selection provides a general method for identifying critical residues involved in DNA-protein interactions. Challenge phage selections have been used to genetically dissect many different prokaryotic and eukaryotic DNA-binding interactions.
Assuntos
Bacteriófagos/genética , Ácidos Nucleicos/metabolismo , Proteínas/metabolismo , Bacteriófago P22/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Lisogenia , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
Mud-P22 derivatives are hybrids between phage Mu and P22 that can be inserted at essentially any desired site on the Salmonella chromosome (Benson and Goldman, 1992; Youderian et al., 1988). Induction of Mud-P22 insertions yields phage particles that, as a population, carry chromosomal DNA from the region between 150 and 250Kb on one side of the insertion. Thus, phage lysates from a representative set of Mud-P22 insertions into the S. typhimurium chromosome yield an ordered library of DNA that provides powerful tools for the genetic and physical analysis of the Salmonella genome. Although Mud-P22 has not yet been used in other species, this approach should be applicable in a variety of other bacteria as well.
Assuntos
Fagos de Salmonella/genética , Salmonella typhimurium/genética , Bacteriófago P22/genética , Bacteriófago mu/genética , Cromossomos Bacterianos/genética , Biblioteca Gênica , Genoma Bacteriano , Mutagênese InsercionalRESUMO
As the number of completed genome sequences increases, there is increasing emphasis on comparative genomic analysis of closely related organisms. Comparison of the similarities and differences between the five publicly available Salmonella genome sequences reveals extensive sequence conservation among the Salmonella serovars. However, horizontal gene transfer has provided each genome with between 10% and 12% of unique DNA. Genome comparisons of the closely related salmonellae emphasize the insights that can be gleaned from sequencing genomes of a single species.
Assuntos
Genoma Bacteriano , Salmonella/genética , Variação Antigênica , Plasmídeos , Salmonella/imunologia , Salmonella/patogenicidade , Homologia de Sequência , VirulênciaRESUMO
Salmonella enterica serovar Enteritidis is a leading cause of food poisoning in the USA and Europe. Although Salmonella serovars share many fimbrial operons, a few fimbriae are limited to specific Samonella serovars. SEF14 fimbriae are restricted to group D Salmonella and the genes encoding this virulence factor were acquired relatively recently. Genomic, genetic and gene expression studies have been integrated to investigate the ancestry, regulation and expression of the sef genes. Genomic comparisons of the Salmonella serovars sequenced revealed that the sef operon is inserted in leuX in Salmonella Enteritidis, Salmonella Paratyphi and Salmonella Typhi, and revealed the presence of a previously unidentified 25 kb pathogenicity island in Salmonella Typhimurium at this location. Salmonella Enteritidis contains a region of homology between the Salmonella virulence plasmid and the chromosome downstream of the sef operon. The sef operon itself consists of four co-transcribed genes, sefABCD, and adjacent to sefD there is an AraC-like transcriptional activator that is required for expression of the sef genes. Expression of the sef genes was optimal during growth in late exponential phase and was repressed during stationary phase. The regulation was coordinated by the RpoS sigma factor.