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Construction and use of a broad-host-range plasmid expressing the lamB gene for utilization of bacteriophage lambda vectors in the marine bacterium Vibrio harveyi
Get PDFThe remarkable success of Escherichia coli as a model organism in molecular genetics was dependent, among other things, on its susceptibility to genetic manipulation. Many versatile and sophisticated genetic tools for molecular biology studies are derived from bacteriophage lambda. However, this bacteriophage is specific for E. coli, and thus lambda-based techniques have been restricted to this bacterium. Plasmids expressing the E. coli gene coding for bacteriophage lambda receptor were reported previously, and introduction of such plasmids into cells of some other bacteria made them sensitive to phage lambda infection. However, we found that these systems were not efficient for Vibrio harveyi, one of the most frequently investigated species of marine bacteria. Here we describe construction of a broad-host-range plasmid expressing the lamB gene. Introduction of this plasmid to V. harveyi cells and expression of lamB made this strain susceptible to bacteriophage lambda adsorption and lambda DNA injection. Foreign genetic material could be introduced into cells of this strain using a cosmid vector
An introduction to DNA chips: principles, technology, applications and analysis
Get PDFThis review describes the recently developed GeneChip technology that provides efficient access to genetic information using miniaturised, high-density arrays of DNA
or oligonucleotide probes. Such microarrays 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
Regulation of the switch from early to late bacteriophage lambda DNA replication
Get PDFThere are two modes of bacteriophage k DNA replication following infection of its host, Escherichia coli. Early after infection, replication occurs according to the theta (teta or circle-to-circle) mode, and is later switched to the sigma (sigma or rolling-circle) mode. It is not known how this switch, occurring at a specific time in the infection cycle, is regulated. Here it is demonstrated that in wildtype cells the replication starting from orik proceeds both bidirectionally and unidirectionally, whereas in bacteria devoid of a functional DnaA protein,
replication from orik is predominantly unidirectional. The regulation of directionality of replication from orik is mediated by positive control of lambda pR promoter activity by DnaA, since the mode of replication of an artificial lambda replicon bearing the ptet promoter instead of pR was found to be independent of DnaA function. These findings and results of density-shift experiments suggest that in dnaA mutants infected with lambda, phage DNA replication proceeds predominantly according to the unidirectional teta mechanism and is switched early after infection to the sigma mode. It is proposed that in wild-type E. coli cells infected with lambda, phage DNA replication proceeds according to a bidirectional teta mechanism early after infection due to efficient transcriptional activation of ori lambda, stimulated by the host DnaA protein. After a few rounds of this type of replication, the resulting increased copy number of teta genomic DNA may cause a depletion of free DnaA protein because of its interaction with the multiple DnaA-binding sites in lambda DNA. It is proposed that this may lead to inefficient transcriptional activation of orik resulting in unidirectional teta replication followed by sigma type replication
ClpP/ClpX-mediated degradation of the bacteriophage λ O protein and regulation of λ phage and λ plasmid replication
Get PDFThe O protein is a replication initiator that binds
to the oriλ region and promotes assembly of the bacteriophage λ replication complex. This protein, although protected from proteases by other elements of the replication complex, in a free form is rapidly degraded in the host, Escherichia coli, by the ClpP/ClpX protease. Nevertheless, the physiological role of this rapid degradation remains unclear. Here we demonstrate that the copy number of plasmids derived from bacteriophage λ is significantly higher in wild-type cells growing in rich media than in slowly growing bacteria. However, λ plasmid copy number in bacteria devoid of the ClpP/ClpX protease was not dependent on the bacterial growth rate and in all minimal media tested was comparable to that observed in wildtype cells growing in a rich medium. Contrary to λ plasmid replication, the efficiency of lytic growth of bacteriophage λ was found to be dependent on the host growth
rate in both wild-type bacteria and clpP and clpX mutants. The activities of two major λ promoters operating during the lytic development, pR and pL, were found to be slightly dependent on the host growth rate. However, when pR activity was significantly decreased in the dnaA mutant, production of phage progeny was completely abolished at low growth rates. These results indicate that the O protein (whose level in E. coli cells depends on the activity of ClpP/ClpX protease) is a major limiting factor in the regulation of λ plasmid replication at low bacterial growth
rates. However, this protein seems to be only one of the
limiting factors in the bacteriophage λ lytic development
under poor growth conditions of host cells. Therefore, it seems that the role of the rapid ClpP/ClpX-mediated proteolysis of the O protein is to decrease the efficiency of early DNA replication of the phage in slowly growing
host cells
DnaA-stimulated transcriptional activation of orilambda: Escherichia coli RNA polymerase beta subunit as a transcriptional activator contact site
Get PDFWe present evidence that Escherichia coli RNA polymerase beta subunit may be a transcriptional activator contact site. Stimulation of the activity of the pR promoter by DnaA protein is necessary for replication of plasmids derived from bacteriophage lambda. We found that DnaA activates the pR promoter in vitro. Particular mutations in the rpoB gene were able to suppress negative effects that certain dnaA mutations had on the replication of lambda plasmids; this suppression was allele-specific. When a potential DnaA-binding sequence located several base pairs downstream of the pR promoter was scrambled by in vitro mutagenesis, the pR promoter was no longer activated by DnaA both in vivo and in vitro. Therefore, we conclude that DnaA may contact the beta subunit of RNA polymerase during activation of the pR promoter. A new classification of prokaryotic transcriptional activators is proposed
Molecular mechanism of heat shock-provoked disassembly of the coliphage lambda replication complex
Get PDFWe have found previously that, in contrast to the free O initiator protein of lambda phage or plasmid rapidly degraded by the Escherichia coli ClpP/ClpX protease, the lambdaO present in the replication complex (RC) is protected from proteolysis. However, in cells growing in a complete medium, a temperature shift from 30 to 43 degrees C resulted in the decay of the lambdaO fraction, which indicated disassembly of RC. This process occurred due to heat shock induction of the groE operon, coding for molecular chaperones of the Hsp60 system. Here we demonstrate that an increase in the cellular concentration of GroEL and GroES proteins is not in itself sufficient to cause RC disassembly. Another requirement is a DNA gyrase-mediated negative resupercoiling of lambda plasmid DNA, which counteracts DNA relaxation and starts to dominate 10 min after the temperature upshift. We presume that RC dissociates from lambda DNA during the negative resupercoiling, becoming susceptible to the subsequent action of GroELS and ClpP/ClpX proteins. In contrast to lambda cro+, in lambda cro- plasmid-harboring cells, the RC reveals heat shock resistance. After temperature upshift of the lambda crots plasmid-harboring cells, a Cro repressor-independent control of lambda DNA replication and heat shock resistance of RC are established before the period of DNA gyrase-mediated negative supercoiling. We suggest that the tight binding of RC to lambda DNA is due to interaction of RC with other DNA-bound proteins, and is related to the molecular basis of the lambda cro- plasmid replication contro
The cbpA chaperone gene function compensates for dnaJ in lambda plasmid replication during amino acid starvation of Escherichia coli
Get PDFWe found previously that lambda plasmid DNA replication in amino acid-starved Escherichia coli relA mutants (i.e., during the relaxed response), which is carried out by the inherited replication complex, is dependent on functions of DnaK and GrpE molecular chaperones but proceeds in a dnaj mutant at a nonpermissive temperature. Here we demonstrate that this replication is inhibited when functions of both dnaJ and cbpA are impaired. In complete media, the growth of the lambda pi A66 phage (capable of replicating in E. coli dnaJ, dnaK, and grpE missense mutants at 30 degrees C), as well as efficiency of transformation by the lambda pi A66 plasmid, is significantly decreased in a dnaJ259 cbpA::kan double mutant. These results strengthen the proposal of other authors (C. Ueguchi, M. Kakeda, H. Yamada, and T. Mizuno, Proc. Natl. Acad. Sci. USA 91:1054-1058, 1994; C. Ueguchi, T. Shiozawa, M. Kakeda, H. Yamada, and T. Mizuno, J. Bacteriol. 177:3894-3896, 1995; and T. Yamashino, M. Kakeda, C. Ueguchi, and T. Mizuno, Mol. Microbiol. 13:475-483, 1994) that the cbpA gene product is a functional analog of the DnaJ chaperone in E. coli
The roles of PCNA-SUMOylation, Mms2-Ubc13 and Rad5 in translesion 10 DNA synthesis in Saccharomyces cerevisiae.
Get PDFBiology, systematics, and clinical manifestations of Zygomycota infections
Get PDFFungi cause opportunistic, nosocomial, and community-acquired infections. Among fungal infections (mycoses) zygomycoses are exceptionally severe, with a mortality rate exceeding 50 %. Immunocompromised hosts, transplant recipients, and diabetic patients with uncontrolled keto-acidosis and high iron serum levels are at risk. Zygomycota are capable of infecting hosts immune to other filamentous fungi. The infection often follows a progressive pattern, with angioinvasion and metastases. Moreover, current antifungal therapy frequently has an unfavorable outcome. Zygomycota are resistant to some of the routinely used antifungals, among them azoles (except posaconazole) and echinocandins. The typical treatment consists of surgical debridement of the infected tissues accompanied by amphotericin B administration. The latter has strong nephrotoxic side effects, which make it unsuitable for prophylaxis. Delayed administration of amphotericin and excision of mycelium-containing tissues worsens survival prognoses. More than 30 species of Zygomycota are involved in human infections, among them Mucorales is the most abundant. Prognosis and treatment suggestions differ for each species, which makes fast and reliable diagnosis essential. Serum sample PCR-based identification often gives false-negative results; culture-based identification is time-consuming and not always feasible. With the dawn of Zygomycota sequencing projects significant advancement is expected, as in the case of treatment of Ascomycota infections
Dataset (sourdough and bread analysis) "Developing lactic acid bacteria starter cultures for wholemeal rye flour bread with improved functionality, nutritional value, taste, appearance and safety"
Get PDFDataset including:
- Chemical composition
- Organic acids
- Phytate
- Alcohols and sugars
- Quality
- Texture and moisture
- Organoleptic assesment
- Active and potential acidity changes during sourdough fermentatio