Biomedical subjects
Publications and source records attributed to Bernd Appel.
The determination of phenotypic and genotypic traits of a group of closely related Vibrio strains from the Baltic Sea did not allow species designation. DNA-DNA hybridization and fatty acid analysis revealed them as Vibrio navarrensis. Therefore we suggest these Vibrios to represent a new biotype, named V. navarrensis biotype pommerensis.
BACKGROUND: Coxiella burnetii, the bacterium causing Q fever, is an obligate intracellular biosafety level 3 agent. Detection and quantification of these bacteria with conventional methods is time consuming and dangerous. During the last years, several PCR based diagnostic assays were developed to detect C. burnetii DNA in cell cultures and clinical samples. We developed and evaluated TaqMan-based real-time PCR assays that targeted the singular icd (isocitrate dehydrogenase) gene and the transposase of the IS1111a element present in multiple copies in the C. burnetii genome. RESULTS: To evaluate the precision of the icd and IS1111 real-time PCR assays, we performed different PCR runs with independent DNA dilutions of the C. burnetii Nine Mile RSA493 strain. The results showed very low variability, indicating efficient reproducibility of both assays. Using probit analysis, we determined that the minimal number of genome equivalents per reaction that could be detected with a 95% probability was 10 for the icd marker and 6.5 for the IS marker. Plasmid standards with cloned icd and IS1111 fragments were used to establish standard curves which were linear over a range from 10 to 10(7) starting plasmid copy numbers. We were able to quantify cell numbers of a diluted, heat-inactivated Coxiella isolate with a detection limit of 17 C. burnetii particles per reaction. Real-time PCR targeting both markers was performed with DNA of 75 different C. burnetii isolates originating from all over the world. Using this approach, the number of IS1111 elements in the genome of the Nine Mile strain was determined to be 23, close to 20, the number revealed by genome sequencing. In other isolates, the number of IS1111 elements varied widely (between seven and 110) and seemed to be very high in some isolates. CONCLUSION: We validated TaqMan-based real-time PCR assays targeting the icd and IS1111 markers of C. burnetii. The assays were shown to be specific, highly sensitive and efficiently reproducible. Cell numbers in dilutions of a C. burnetii isolate were reliably quantified. PCR quantification suggested a high variability of the number of IS1111 elements in different C. burnetii isolates, which may be useful for further phylogenetic studies.
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.
We evaluated real-time PCR assays for the detection of C. burnetii which targets sequences that are present either in one (icd) or in several copies (transposase of IS1111a) on the chromosome. The assays are highly sensitive, with reproducible detection limits of approximately 10 copies per reaction, at least 100 times more sensitive than capture ELISA, when performed on infected placenta material and specific for C. burnetii. The numbers of IS1111 elements in the genomes of 75 C. burnetii isolates were quantified by real-time PCR and proved to be highly variable.
Bdellovibrio bacteriovorus is a predatory bacterium that grows and replicates within the periplasm of a large variety of Gram-negative bacteria. So far, the host-interaction locus (hit locus) is the only genetic locus that is implicated in the obligate predatory lifestyle. Sequence analysis revealed that upstream of the hit locus, the genomic regions of the two obligate predatory B. bacteriovorus-type strains HD100 and HD114 encode genes for pilus formation. As pili might be involved in the invasion process, the transcriptional activity of the hit locus and of a putative pilin gene (flp1) of the pilus cluster were studied in synchronized cultures of B. bacteriovorus with Escherichia coli K-12 as prey bacteria. Stages of the life cycle were monitored with scanning electronic microscopy and transcriptional analyses were performed by quantitative reverse transcription polymerase chain reaction. Our study revealed an increased expression level of the putative hit and flp1 genes in the attack phase of B. bacteriovorus, whereas the transcriptional activity significantly decreased during the intracellular replication phase.
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.
Bdellovibrio-and-like organisms (BALOs) are predatory bacteria that prey upon Gram-negative bacteria and are taxonomically subsumed in the order Bdellovibrionales. Despite their unique lifestyle, these bacteria show remarkable genotypic diversities. The outer membrane of the predators is likely to play an important role during the recognition and invasion stage, as well as in the intraperiplasmic growth phase. In this study, the outer membrane protein fractions of type strains of Bdellovibrio, Bacteriovorax and Peredibacter were investigated, revealing the presence of outer membrane proteins (Omps) similar to the major Omps of Bdellovibrio bacteriovorus. The primary structures of these Omps of Bdellovibrio sp. W, Bacteriovorax stolpii and Peredibacter starrii were elucidated by a combined mass spectrometric-reverse genetic approach. The similarity between the analyzed Omps of the investigated BALOs ranges from 32% to 89% showing conserved amino acid regions in their primary structure.
The genus Mycobacterium comprises highly pathogenic as well as opportunistic or apathogenic species exhibiting a great variability with respect to their ability to persist or multiply within monocytic host cells. The impact of the permeability of the mycobacterial outer membrane on intracellular persistence was studied. For this purpose, a Mycobacterium smegmatis mutant with a deletion of the major porin gene mspA and a second mutant lacking mspA and the homologous porin gene mspC were used. Deletion of mspA together with mspC significantly enhanced intracellular persistence in murine bone marrow macrophages, the mouse macrophage cell line J774A.1 and Acanthamoeba castellanii. Complementation of mspA in the porin mutant strains resulted in restoration of the wild-type phenotype with respect to intracellular persistence. This is the first report to show that the deletion of porins of mycobacteria results in improved persistence in eukaryotic cells, demonstrating that the intracellular persistence of M. smegmatis depends upon the permeability of the outer membrane.
The Yersinia enterocolitica prophage PY54 replicates as a linear DNA molecule with covalently closed ends. For replication of a circular PY54 minimal replicon that has been derived from a linear minireplicon, two phage-encoded loci are essential in Escherichia coli: (i) the reading frame of the replication initiation gene repA and (ii) its 212-bp origin located within the 3' portion of repA. The RepA protein acts in trans on the origin since we have physically separated the PY54 origin and repA onto a two-plasmid origin test system. For this trans action, the repA 3' end carrying the origin is dispensable. Mutagenesis by alanine scan demonstrated that the motifs for primase and for nucleotide binding present in the protein are essential for RepA activity. The replication initiation functions of RepA are replicon specific. The replication initiation proteins DnaA, DnaG, and DnaB of the host are unable to promote origin replication in the presence of mutant RepA proteins that carry single residue exchanges in these motifs. The proposed origins of the known related hairpin prophages PY54, N15, and PKO2 are all located toward the 3' end of the corresponding repA genes, where several structure elements are conserved. Origin function depends on the integrity of these elements.
Mycobacteria are characterized by an extremely thick hydrophobic cell wall restricting the permeability for small hydrophilic compounds. Recently, a new efficient porin (MspA) has been identified in Mycobacterium smegmatis, which is lacking in slow-growing mycobacteria. Since we were interested in investigating the influence of porins on growth of slow-growing Mycobacterium bovis BCG, we inserted a 3429 bp DNA fragment from M. smegmatis carrying the mspA gene in an integrative vector and transferred it into M. bovis BCG. Expression of mspA in the BCG derivative was shown by RT-PCR and Western blot. Quantification of bacterial growth on agar plates demonstrated two- to four-fold better growth of the BCG derivative with the transferred DNA compared with the reference strain. Transposon mutagenesis proved the mspA gene to be responsible for the growth enhancement. Intracellular multiplication of the BCG derivative in the mouse macrophage cell line J774 and the human pneumocyte cell line A549 was also clearly enhanced pointing to a possible role of porins in the interaction of mycobacteria with their hosts.
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.
Bdellovibrio bacteriovorus is a predatory bacterium that is capable of invading a number of gram-negative bacteria. The life cycle of this predator can be divided into a nonreproductive phase outside the prey bacteria and a multiplication phase in their periplasm. It was suggested that during the reproduction phase, B. bacteriovorus reutilizes unmodified components of the prey's cell wall. We therefore examined the outer membranes of B. bacteriovorus strains HD100 (DSM 50701) and HD114 (DSM 50705) by using Escherichia coli, Yersinia enterocolitica, and Pseudomonas putida as prey organisms. The combined sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometric analyses revealed novel and innate major outer membrane proteins (OMPs) of B. bacteriovorus strains. An incorporation of prey-derived proteins into the cell wall of B. bacteriovorus was not observed. The corresponding genes of the B. bacteriovorus strains were elucidated by a reverse-genetics approach, and a leader peptide was deduced from the gene sequence and confirmed by Edman degradation. The host-independent mutant strain B. bacteriovorus HI100 (DSM 12732) growing in the absence of prey organisms possesses an OMP similar to the major OMPs of the host-dependent strains. The similarity of the primary structure of the OMPs produced by the three Bdellovibrio strains is between 67 and 89%. The leader peptides of all OMPs have a length of 20 amino acids and are highly conserved. The molecular sizes of the mature proteins range from 34.9 to 37.6 kDa. Secondary-structure predictions indicate preferential alpha-helices and little beta-barrel structures.
A group of hemolytic Vibrio strains was isolated from surface water of the Baltic Sea in 1995. A typical representative strain, CH-291, was found to lyse washed human and animal erythrocytes. Hemolysis was found to be calcium-dependent and occurred over a temperature range from 25 to 37 degrees C. The hemolysin-encoding genes were identified by screening a genomic library of total DNA from strain CH-291. A cloned chromosomal DNA fragment of 15.6 kb conferred to Escherichia coli DH5alpha a hemolytic phenotype. Hybridization and sequence analysis showed the cloned sequence to be unique to these Baltic Sea Vibrio isolates and therefore provides a useful marker for their identification. Moreover, the cloned 15.6-kb DNA fragment possessed structural features typical for genetic islands, including a decreased GC content and a flanking cryptic insertion sequence element.
The temperate Yersinia phage PY54 belongs to the unusual group of phages that replicate as linear plasmids with covalently closed ends. Besides Escherichia coli phage N15, PY54 is the only member of this group to be identified. We have determined the complete sequence (46,339 bp) of the PY54 genome. Bioinformatic analyses revealed 67 open reading frames (ORFs) with good coding potential located on both DNA strands. The comparison of the deduced PY54 gene products with known proteins encoded by other phages and bacteria along with functional studies have enabled us to assign the possible functions of 25 ORFs. In the left arm of the PY54 genome, we identified a number of ORFs that obviously code for head and tail proteins. Furthermore, this part of the phage genome contains genes probably involved in plasmid partitioning. Regarding the predicted gene functions and gene order, the PY54 and N15 left arms are similar. However, there are only weak DNA homologies and, in contrast to N15, the Yersinia phage harbours only a few ORFs related to genes found in lambdoid phages. The PY54 right arm comprises mainly regulatory genes as well as genes important for plasmid replication, DNA methylation, and host cell lysis. Out of 36 deduced products of the right arm, 13 revealed strongest database homologies to N15 proteins, of which the protelomerase and the Rep protein are exclusively homologous to their N15 counterparts. A number of PY54 genes essential for the lytic or lysogenic cycle were identified by functional analysis and characterization of phage mutants. In order to study transcription during the lytic and lysogenic stage, we analysed 34 PY54 ORFs by reverse transcriptase (RT)-PCR. The phage transcription patterns in lysogenic bacteria and at the late lytic stage of infection are nearly identical. The reasons for this finding are spontaneous release of phages during lysogeny and a high rate of phages that lysogenize their Yersinia host upon infection.
Bdellovibrio bacteriovorus are predatory bacteria that penetrate Gram-negative bacteria and grow intraperiplasmically at the expense of the prey. It was suggested that B. bacteriovorus partially degrade and reutilize lipopolysaccharide (LPS) of the host, thus synthesizing an outer membrane containing structural elements of the prey. According to this hypothesis a host-independent mutant should possess a chemically different LPS. Therefore, the lipopolysaccharides of B. bacteriovorus HD100 and its host-independent derivative B. bacteriovorus HI100 were isolated and characterized by SDS-polyacrylamide gel electrophoresis, immunoblotting, and mass spectrometry. LPS of both strains were identified as smooth-form LPS with different repeating units. The lipid As were isolated after mild acid hydrolysis and their structures were determined by chemical analysis, by mass spectrometric methods, and by NMR spectroscopy. Both lipid As were characterized by an unusual chemical structure, consisting of a beta-(1-->6)-linked 2,3-diamino-2,3-dideoxy-d-glucopyranose disaccharide carrying six fatty acids that were all hydroxylated. Instead of phosphate groups substituting position O-1 of the reducing and O-4' of the nonreducing end alpha-d-mannopyranose residues were found in these lipid As. Thus, they represent the first lipid As completely missing negatively charged groups. A reduced endotoxic activity as determined by cytokine induction from human macrophages was shown for this novel structure. Only minor differences with respect to fatty acids were detected between the lipid As of the host-dependent wild type strain HD100 and for its host-independent derivative HI100. From the results of the detailed analysis it can be concluded that the wild type strain HD100 synthesizes an innate LPS.
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