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Identification of pathogenic microbial cells and spores by electrochemical detection on a biochip
Get PDFBACKGROUND:
Bacillus cereus constitutes a significant cause of acute food poisoning in humans. Despite the recent development of different detection methods, new effective control measures and better diagnostic tools are required for quick and reliable detection of pathogenic micro-organisms. Thus, the objective of this study was to determine a simple method for rapid identification of enterotoxic Bacillus strains. Here, a special attention is given to an electrochemical biosensor since it meets the requirements of minimal size, lower costs and decreased power consumption.
RESULTS:
A bead-based sandwich hybridization system was employed in conjugation with electric chips for detection of vegetative cells and spores of Bacillus strains based on their toxin-encoding genes. The system consists of a silicon chip based potentiometric cell, and utilizes paramagnetic beads as solid carriers of the DNA probes. The specific signals from 20 amol of bacterial cell or spore DNA were achieved in less than 4 h. The method was also successful when applied directly to unpurified spore and cell extract samples. The assay for the haemolytic enterotoxin genes resulted in reproducible signals from B. cereus and B. thuringiensis while haemolysin-negative B. subtilis strain did not yield any signal.
CONCLUSIONS:
The sensitivity, convenience and specificity of the system have shown its potential. In this respect an electrochemical detection on a chip enabling a fast characterization and monitoring of pathogens in food is of interest. This system can offer a contribution in the rapid identification of bacteria based on the presence of specific genes without preceding nucleic acid amplification
An introduction to DNA chips
Get PDFThe GeneChip, which is gaining popularity throughout the field of molecular biology and biotechnology, is a recently developed technique which provides efficient access to genetic information using miniaturized, high-density arrays of DNA or oligonucleotide probes. Both fundamental and applied research benefit greatly from the specific advantages of this method. Initially developed to enhance
genome sequencing projects, the technology is rapidly evolving and has been adapted to a large set of applications. DNA chips are powerful tools to study
the molecular basis of interactions on a scale that would be impossible using conventional analysis. The recent development of the microarray technology has greatly accelerated the investigation of gene regulation. Arrays are mostly used to identify which genes are turned on or off in a cell or tissue, and also to evaluate the
extent of a gene’s expression under various conditions. Indeed, this technology has been successfully applied to investigate simultaneous expression of many thousands
of genes and to the detection of mutations or polymorphisms, as well as for their mapping and sequencing. In this chapter, we discuss the format of DNA chips, the technology used to fabricate and read them, and their applications
Non-random distribution of GATC sequences in regions of promoters stimulated by the SeqA protein of Escherichia coli
Get PDFThe SeqA protein of Escherichia coli is not only the main negative regulator of DNA replication initiation but also a specific transcription factor. It binds to hemimethylated GATC sequences and, with somewhat different specificity, to fully methylated GATC regions. Recently, a microarray analysis was reported, in which transcriptomes of wild-type and ΔseqA strains were compared. Although in the seqA mutant the levels of some transcripts were significantly decreased while certain transcripts were evidently more abundant relative to wild-type bacteria, no correlation between the presence of GATC motifs in promoter sequences and transcription activity was found. However, here we show that when larger DNA fragments, encompassing positions from –250 to +250 relative to the transcription start site, are analyzed, some common features of GATC distribution near the promoters activated by SeqA can be demonstrated. Nevertheless, it seems that the GATC pattern is not the only determinant of SeqA-dependence of promoter activity
UV- and MMS-induced mutagenesis of lambdaO(am)8 phage under nonpermissive conditions for phage DNA replication.
Get PDFMutagenesis in Escherichia coli, a subject of many years of study is considered to be related to DNA replication. DNA lesions nonrepaired by the error-free nucleotide excision repair (NER), base excision repair (BER) and recombination repair (RR), stop replication at the fork. Reinitiation needs translesion synthesis (TLS) by DNA polymerase V (UmuC), which in the presence of accessory proteins, UmuD', RecA and ssDNA-binding protein (SSB), has an ability to bypass the lesion with high mutagenicity. This enables reinitiation and extension of DNA replication by DNA polymerase III (Pol III). We studied UV- and MMS-induced mutagenesis of lambdaO(am)8 phage in E. coli 594 sup+ host, unable to replicate the phage DNA, as a possible model for mutagenesis induced in nondividing cells (e.g. somatic cells). We show that in E. coli 594 sup+ cells UV- and MMS-induced mutagenesis of lambdaO(am)8 phage may occur. This mutagenic process requires both the UmuD' and C proteins, albeit a high level of UmuD' and low level of UmuC seem to be necessary and sufficient. We compared UV-induced mutagenesis of lambdaO(am)8 in nonpermissive (594 sup+) and permissive (C600 supE) conditions for phage DNA replication. It appeared that while the mutagenesis of lambdaO(am)8 in 594 sup+ requires the UmuD' and C proteins, which can not be replaced by other SOS-inducible protein(s), in C600 supE their functions may be replaced by other inducible protein(s), possibly DNA polymerase IV (DinB). Mutations induced under nonpermissive conditions for phage DNA replication are resistant to mismatch repair (MMR), while among those induced under permissive conditions, only about 40% are resistant
Stress responses and replication of plasmids in bacterial cells
Get PDFPlasmids, DNA (or rarely RNA) molecules which replicate in cells autonomously (independently of chromosomes) as non-essential genetic elements, play important roles for microbes grown under specific environmental conditions as well as in scientific laboratories and in biotechnology. For example, bacterial plasmids are excellent models in studies on regulation of DNA replication, and their derivatives are the most commonly used vectors in genetic engineering. Detailed mechanisms of replication initiation, which is the crucial process for efficient maintenance of plasmids in cells, have been elucidated for several plasmids. However, to understand plasmid biology, it is necessary to understand regulation of plasmid DNA replication in response to different environmental conditions in which host cells exist. Knowledge of such regulatory processes is also very important for those who use plasmids as expression vectors to produce large amounts of recombinant proteins. Variable conditions in large-scale fermentations must influence replication of plasmid DNA in cells, thus affecting the efficiency of recombinant gene expression significantly. Contrary to extensively investigated biochemistry of plasmid replication, molecular mechanisms of regulation of plasmid DNA replication in response to various environmental stress conditions are relatively poorly understood. There are, however, recently published studies that add significant data to our knowledge on relations between cellular stress responses and control of plasmid DNA replication. In this review we focus on plasmids derived from bacteriophage λthat are among the best investigated replicons. Nevertheless, recent results of studies on other plasmids are also discussed shortly
Cloning and characterization of the first member of the Nudix family from Arabidopsis thaliana.
Get PDFThe sequence motif commonly called a Nudix box, represented by (GX(5)EX(7)REVXEEXGU) is the marker of a widely distributed family of enzymes that catalyze the hydrolysis of a variety of nucleoside diphosphate derivatives. Here we describe the cloning and characterization of an Arabidopsis thaliana cDNA encoding a Nudix hydrolase that degrades NADH. The deduced amino acid sequence of AtNUDT1 contains 147 amino acids. The recombinant AtNUDT1 was expressed in Escherichia coli and purified. In the presence of Mn(2+) and the optimal pH of 7. 0, the recombinant AtNUDT1 catalyzed the hydrolysis of NADH with a K(m) value of 0. 36 mm. A V(max) of 12. 7 units mg (-1) for NADH was determined. The recombinant AtNUDT1 migrated as a dimer on a gel filtration column. Biochemical analysis of recombinant AtNUDT1 indicated that the first characterized member of the Nudix family from A. thaliana is a NADH pyrophosphatase
How do marine bacteria produce light, why are they luminescent, and can we employ bacterial bioluminescence in aquatic biotechnology?
Get PDFBioluminescence, the phenomenon of light production by living organisms, occurs in forms of life as various as bacteria, fungi and animals. Nevertheless, light-emitting bacteria are the most abundant and widespread of luminescent organisms. Interestingly, most species of such bacteria live in marine environments. In this article, the biochemical mechanism of bacterial luminescence and its genetic regulation are summarized. Although the biochemistry and genetics of light emission by cells have been investigated in detail, the biological role of bacterial luminescence has remained obscure. Here, we discuss recent discoveries that shed new light on this problem. Finally, we provide examples of how bacterial luminescence can be employed in marine biotechnology, especially in the detection of toxic and mutagenic pollution in aquatic environments
Composition of the λ plasmid heritable replication complex
No full textPrevious studies indicated during replication of plasmids derived from bacteriophage λ (the so-called λ plasmids), that, once assembled, replication complex can be inherited by one of the two daughter plasmid copies after each replication round, and may function in subsequent replication rounds. It seems that similar processes occur during replication of other DNA molecules, including chromosomes of the yeast Saccharomyces cerevisiae. However, apart from some suggestions based on genetic experiments, composition of the λ heritable replication complex remains unknown. In amino acid-starved Escherichia coli relA mutants, replication of λ plasmid DNA is carried out exclusively by the heritable replication complex as assembly of new complexes is impaired due to inhibition of protein synthesis. Here, using a procedure based on in vivo cross-linking, cell lysis, immunoprecipitation with specific sera, de-cross-linking and PCR analysis, we demonstrate that the λ heritable replication complex consists of O, P, DnaB and, perhaps surprisingly, DnaK proteins
Inhibition of spontaneous induction of lambdoid prophages in Escherichia coli cultures: simple procedures with possible biotechnological applications
Get PDFBACKGROUND:
Infections of bacterial cultures by bacteriophages are serious problems in biotechnological laboratories. Apart from such infections, prophage induction in the host cells may also be dangerous. Escherichia coli is a commonly used host in biotechnological production, and many laboratory strains of this bacterium harbour lambdoid prophages. These prophages may be induced under certain conditions leading to phage lytic development. This is fatal for further cultivations as relatively low, though still significant, numbers of phages may be overlooked. Thus, subsequent cultures of non-lysogenic strains may be infected and destroyed by such phage.
RESULTS:
Here we report that slow growth of bacteria decreases deleterious effects of spontaneous lambdoid prophage induction. Moreover, replacement of glucose with glycerol in a medium stimulates lysogenic development of the phage after infection of E. coli cells. A plasmid was constructed overexpressing the phage 434 cI gene, coding for the repressor of phage promoters which are necessary for lytic development. Overproduction of the cI repressor abolished spontaneous induction of the lambda(imm434) prophage.
CONCLUSIONS:
Simple procedures that alleviate problems with spontaneous induction of lambdoid prophage and subsequent infection of E. coli strains by these phages are described. Low bacterial growth rate, replacement of glucose with glycerol in a medium and overproduction of the cI repressor minimise the risk of prophage induction during cultivation of lysogenic bacteria and subsequent infection of other bacterial strains
