Biomedical subjects

Daniela Jacob

Publications and source records attributed to Daniela Jacob.

4 recordsLinked to original sources

PubMed2006

Characterization of Bacillus anthracis-like bacteria isolated from wild great apes from Cote d'Ivoire and Cameroon.

We present the microbiological and molecular characterization of bacteria isolated from four chimpanzees and one gorilla thought to have died of an anthrax-like disease in Côte d'Ivoire and Cameroon. These isolates differed significantly from classic Bacillus anthracis by the following criteria: motility, resistance to the gamma phage, and, for isolates from Cameroon, resistance to penicillin G. A capsule was expressed not only after induction by CO(2) and bicarbonate but also under normal growth conditions. Subcultivation resulted in beta-hemolytic activity and gamma phage susceptibility in some subclones, suggesting differences in gene regulation compared to classic B. anthracis. The isolates from Côte d'Ivoire and Cameroon showed slight differences in their biochemical characteristics and MICs of different antibiotics but were identical in all molecular features and sequences analyzed. PCR and Southern blot analyses confirmed the presence of both the toxin and the capsule plasmid, with sizes corresponding to the B. anthracis virulence plasmids pXO1 and pXO2. Protective antigen was expressed and secreted into the culture supernatant. The isolates possessed variants of the Ba813 marker and the SG-749 fragment differing from that of classic B. anthracis strains. Multilocus sequence typing revealed a close relationship of our atypical isolates with both classic B. anthracis strains and two uncommonly virulent Bacillus cereus and Bacillus thuringiensis isolates. We propose that the newly discovered atypical B. anthracis strains share a common ancestor with classic B. anthracis or that they emerged recently by transfer of the B. anthracis plasmids to a strain of the B. cereus group.

Alleles↗

PubMed2005

Viral promoters can initiate expression of toxin genes introduced into Escherichia coli.

BACKGROUND: The expression of recombinant proteins in eukaryotic cells requires the fusion of the coding region to a promoter functional in the eukaryotic cell line. Viral promoters are very often used for this purpose. The preceding cloning procedures are usually performed in Escherichia coli and it is therefore of interest if the foreign promoter results in an expression of the gene in bacteria. In the case molecules toxic for humans are to be expressed, this knowledge is indispensable for the specification of safety measures. RESULTS: We selected five frequently used viral promoters and quantified their activity in E. coli with a reporter system. Only the promoter from the thymidine kinase gene from HSV1 showed no activity, while the polyhedrin promoter from baculovirus, the early immediate CMV promoter, the early SV40 promoter and the 5' LTR promoter from HIV-1 directed gene expression in E. coli. The determination of transcription start sites in the immediate early CMV promoter and the polyhedrin promoter confirmed the existence of bacterial -10 and -35 consensus sequences. The importance of this heterologous gene expression for safety considerations was further supported by analysing fusions between the aforementioned promoters and a promoter-less cytotoxin gene. CONCLUSION: According to our results a high percentage of viral promoters have the ability of initiating gene expression in E. coli. The degree of such heterologous gene expression can be sufficient for the expression of toxin genes and must therefore be considered when defining safety measures for the handling of corresponding genetically modified organisms.

Bacterial Proteins↗

PubMed2004

Yeast DNA sequences initiating gene expression in Escherichia coli.

DNA transfer between pro- and eukaryotes occurs either during natural horizontal gene transfer or as a result of the employment of gene technology. We analysed the capacity of DNA sequences from a eukaryotic donor organism (Saccharomyces cerevisiae) to serve as promoter region in a prokaryotic recipient (Escherichia coli) by creating fusions between promoterless luxAB genes from Vibrio harveyi and random DNA sequences from S. cerevisiae and measuring the luminescence of transformed E. coli. Fifty-four out of 100 randomly analysed S. cerevisiae DNA sequences caused considerable gene expression in E. coli. Determination of transcription start sites within six selected yeast sequences in E. coli confirmed the existence of bacterial -10 and -35 consensus sequences at appropriate distances upstream from transcription initiation sites. Our results demonstrate that the probability of transcription of transferred eukaryotic DNA in bacteria is extremely high and does not require the insertion of the transferred DNA behind a promoter of the recipient genome.

Artificial Gene Fusion↗

PubMed2002

Plant-specific promoter sequences carry elements that are recognised by the eubacterial transcription machinery.

During evolution the promoter elements from prokaryotes and eukaryotes have developed differently with regard to their sequence and structure, implying that in general a transfer of eukaryotic promoter sequences into prokaryotes will not cause an efficient gene expression. However, there have been reports on the functionality of the 35S promoter from cauliflower mosaic virus (CaMV) in bacteria. We therefore decided to experimentally investigate the capability of plant promoter sequences to direct gene expression in various bacteria. Accordingly, we tested ten different plant-specific promoters from Solanum tuberosum, Nicotiana tabacum, CaMV, Agrobacterium tumefaciens, and A. rhizogenes for their ability to initiate transcription in five different eubacterial species (Escherichia coli, Yersinia enterocolitica, A. tumefaciens, Pseudomonas putida, and Acinetobacter sp. BD413). To monitor the strength of the plant-specific promoters in bacteria we created fusions between these promoters and the coding region of the luciferase genes from Vibrio harveyi and measured the luminescence in the bacteria. Heterologous gene expression was observed in 50% of the combinations analysed. We then mapped the transcription start site caused by one of the plant-specific promoters, the ST-LS1 promoter from S. tuberosum, in these bacterial species. The location of the mapped transcription start site indicated that the sequences of the plant promoter themselves were recognised by the bacterial transcription apparatus. The recognition of plant-specific promoter sequences by the bacterial RNA polymerase was further confirmed by site-directed mutagenesis of the ST-LS1 promoter and the analysis of the effects of the mutations on the strength of gene expression in E. coli. Using these mutants in our reporter assays we could localise the sequences of the ST-LS1 promoter serving as -10 region in E. coli. The results of our study show that promoter sequences are much less specific than is generally assumed. This is of great importance for our knowledge about the evolution of gene expression systems and for the construction of optimised expression vectors.

Bacteria↗