Sabine Rölleke

Application of broad-range 16S rRNA PCR amplification and DGGE fingerprinting for detection of tick-infecting bacteria

Ticks play an important role in the transmission of arthropod-borne diseases of viral, protozoal and bacterial origin. The present article describes a molecular-biological based method, which facilitated the broad-range analyses of bacterial communities in ixodid ticks (Ixodes ricinus). DNA was extracted both from single ticks and pooled adult ticks. Eubacterial 16S rRNA gene fragments (16S rDNA) were amplified by polymerase chain reaction (PCR) with broad-range ribosomal primers. Sequences spanning the hypervariable V3 region of the 16S rDNA and representing individual bacterial taxons were separated by denaturing gradient gel electrophoresis (DGGE). For phylogenetic identification, DGGE bands were exised, cloned and sequenced. In addition, we set up a 16S rDNA clone library which was screened by DGGE. Sequences were compared with sequences of known bacteria listed in the GenBank database. A number of bacteria were affiliated with the genera Rickettsia, Bartonella, and Borrelia, which are known to be pathogenic and transmitted by ticks. Two sequences were related to the yet to be cultivated Haemobartonella. To our knowledge, Haemobartonella has never been directly detected in I. ricinus. In addition, members of the genera Staphylococcus, Rhodococcus, Pseudomonas, and Moraxella were detected, which have not been identified in ticks so far. Two bacteria were most closely related to a rickettsial endosymbiont of an Acanthamoeba sp., and to an endosymbiont (Legionellaceae, Coxiella group) of the microarthropod Folsomia candida. The results prove that 16S rDNA genotyping in combination with DGGE analysis is a promising approach for the detection and identification of bacteria infecting ticks, regardless of whether these bacteria are fastidious, obligate intracellular or noncultivable.

An advanced molecular strategy to identify bacterial communities on art objects.

The application of culture-independent techniques based on molecular biological methods, especially on the PCR amplification of 16S rRNA genes, attempts to overcome some shortcomings of conventional cultivation methods and reveals far more complex bacterial communities on art objects than can be shown by cultivation methods. One of the major challenges of investigating microbial growth on art objects by molecular means is the extraction of DNA, due to small sample amounts and PCR inhibitors. In the present study, we introduce a DNA extraction protocol, which allowed the extraction of PCR-amplifiable DNA from samples derived from lime wall paintings and loamy soil underground. The DNA extracts were used to amplify 16S ribosomal fragments, which were subsequently analyzed by denaturing gradient gel electrophoresis (DGGE). In parallel with the DGGE analysis, clone libraries containing PCR fragments of the ribosomal gene were constructed and clones were screened by DGGE. Clone libraries allow the inclusion of the entire 16S rDNA sequence in the phylogenetic analyses of microorganisms, providing a more reliable phylogenetic identification of microorganisms than is obtained from sequence analyses of excised and directly sequenced DGGE bands.

Phylogenetic diversity of bacteria associated with Paleolithic paintings and surrounding rock walls in two Spanish caves (Llonín and La Garma)

Bacterial diversity in caves is still rarely investigated using culture-independent techniques. In the present study, bacterial communities on Paleolithic paintings and surrounding rock walls in two Spanish caves (Llonín and La Garma) were analyzed, using 16S rDNA-based denaturing gradient gel electrophoresis community fingerprinting and phylogenetic analyses without prior cultivation. Results revealed complex bacterial communities consisting of a high number of novel 16S rDNA sequence types and indicated a high biodiversity of lithotrophic and heterotrophic bacteria. Identified bacteria were related to already cultured bacteria (39 clones) and to environmental 16S rDNA clones (46 clones). The nearest phylogenetic relatives were members of the Proteobacteria (41.1%), of the Acidobacterium division (16.5%), Actinobacteria (20%), Firmicutes (10.6%), of the Cytophaga/Flexibacter/Bacteroides division (5.9%), Nitrospira group (3.5%), green non-sulfur bacteria (1.2%), and candidate WS3 division (1.2%). Thirteen of these clones were most closely related to those obtained from the previous studies on Tito Bustillo Cave. The comparison of the present data with the data obtained previously from Altamira and Tito Bustillo Caves revealed similarities in the bacterial community components, especially in the high abundance of the Acidobacteria and Rhizobiaceae, and in the presence of bacteria related to ammonia and sulfur oxidizers.

Comparative analyses of the bacterial diversity on two different biodeteriorated wall paintings by DGGE and 16S rDNA sequence analysis.

Abstract The bacterial diversity associated with two different biodeteriorated wall paintings in Herberstein (Austria) and Greene (Germany) was investigated and compared using a molecular approach combining fingerprinting by DGGE (denaturing gradient gel electrophoresis) with the screening of 16S rDNA clone libraries by DGGE and sequencing. In total, 70 16S rDNA sequences were obtained. Twenty-three sequences were phylogenetically affiliated with genera of the Actinobacteria , namely Arthrobacter , Actinobispora , Amycolata , Asiosporangium , Frankia , Geodermatophilus , Nocardioides , Promicromonospora , Pseudonocardia , Rubrobacter , Streptomonospora , Saccharopolyspora , Sphaerobacter and Thermocrismum . Twenty-seven sequences were affiliated with genera of the Proteobacteria , namely Aquaspirillum , Chromohalobacter , Deleya , Erythrobacter , Halomonas , Porphyrobacter , Pseudomonas , Rhizobium , Salmonella and unidentified γ - Proteobacteria . Nineteen sequences were affiliated with unidentified Cytophagales . One sequence was affiliated with the Chloroflexaceae group. Most genera were present in more than one sample. The bacterial communities present on the two different wall paintings showed only similarities in members of unidentified Cytophagales and of the genera Frankia , Geodermatophilus and Arthrobacter . Cultivation experiments for one sample were carried out in parallel to the molecular approach. Isolates were clustered by FAME (fatty acid methyl ester analysis) and representative members of each cluster were additionally analyzed by DGGE. No similar organisms could be detected by the cultivation approach and the molecular approach. Isolates were phylogenetically affiliated to the genera Bacillus , Paenibacillus , Micrococcus , Staphylococcus , Methylobacterium and Halomonas . The sequence of the isolated Halomonas differed from the Halomonas sequences, which were obtained by the molecular approach. The combined approach of molecular and culturing techniques gives a truer picture of all bacterial organisms on/in a surface than either alone.

Analysis of fungal communities on historical church window glass by denaturing gradient gel electrophoresis and phylogenetic 18S rDNA sequence analysis.

Besides lichens and bacteria, fungi play a crucial role in the biodeterioration of historical glass. In the present paper, the fungal diversity on the surface of two historical church window glasses was investigated by 18S rDNA-based denaturing gradient gel electrophoresis (DGGE) analysis. 566-bp 18S rDNA-specific clone libraries were constructed with primer set NS1/NS2+10. Positive clones were reamplified with primer sets EF4/518rGC (426-bp fragments) and NS26/518rGC (316-bp fragments), amplicons were screened by DGGE and clustered according to their position in DGGE. Results indicated that fungal 18S rDNA clone libraries should be screened with at least two different primer sets to obtain the maximum number of different clones. For phylogenetic sequence analyses, clone inserts were sequenced and compared with 18S rDNA sequences listed in the EMBL database. Similarity values ranged from 93.7% to 99.81% to known fungi. Analyses revealed complex fungal communities consisting of members and relatives of the genera Aspergillus, Aureobasidium, Coniosporum, Capnobotryella, Engyodontium, Geomyces, Kirschsteiniothelia, Leptosphaeria, Rhodotorula, Stanjemonium, Ustilago, and Verticillium. The genera Geomyces and Aureobasidium were present on both glass surfaces. Some genera had not been detected on historical glass so far.

Medieval wall paintings : a habitat for archaea : identification of archaea by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified gene fragments coding for 16S rRNA in a medieval wall painting

Abstract This report describes the detection and identification of archaea in a deteriorated medieval wall painting from the 13th century by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified DNA encoding 16S rRNA without prior cultivation of the organisms. Total DNA was extracted from wall painting material and 547 bp fragments of the 16S rDNA were amplified with archaeal specific primers. Six out of ten analyzed samples showed the presence of archaeal 16S rDNA fragments. The PCR products of uniform length presumably obtained from different archaea species were separated according to their sequence differences by DGGE to visualize the diversity of the archaeal community within the wall painting material. By sequencing an individual DNA band and comparing the data with known 16S rDNA sequences, the corresponding bacteria were identified as members or close relatives of the genus Halobacterium . The results suggest that archaea and, in particular, extremely halophilic species might be more widely involved in deterioration and discolouration processes than commonly recognized.

Analysis of bacterial communities on historical glass by denaturing gradient gel electrophoresis of PCR-amplified gene fragments coding for 16S rRNA.

The present study describes the analysis of bacterial communities on historical window glass by denaturing gradient gel electrophoresis of PCR-amplified 16S rDNA fragments. So far, only a few studies have been published in which the microflora and the corrosion mechanisms of glass surfaces have been investigated. Some microorganisms, especially fungi, have been isolated from different glass samples in the past. However, our results demonstrate that bacterial communities on biodeteriorated glass surfaces are much more complex than previously believed. In addition, bacteria were identified, which have never been isolated from glass samples before.

Rubrobacter-related bacteria associated with rosy discolouration of masonry and lime wall paintings

Abstract. A molecular approach was chosen to analyse the correlation between bacterial colonisation and rosy discolouration of masonry and lime wall paintings of two historically important buildings in Austria and Germany. The applied molecular method included PCR amplification of genes encoding the small subunit rRNA of bacteria (16S rDNA), genetic fingerprinting by denaturing gradient gel electrophoresis (DGGE), construction of 16S rDNA clone libraries, and comparative phylogenetic sequence analyses. The bacterial community of one red-pigmented biofilm sampled in Herberstein (Austria) contained bacteria phylogenetically related to the genera Saccharopolyspora, Nocardioides, Pseudonocardia, Rubrobacter, and to a Kineococcus-like bacterium. The bacterial community of the second red-pigmented biofilm sampled in Herberstein contained bacteria related to Arthrobacter, Comamonas, and to Rubrobacter. Rubrobacter-related 16S rDNA sequences were the most abundant. In the red-pigmented biofilm sampled in Burggen (Germany), only Rubrobacter-related bacteria were identified. No Rubrobacter-related bacteria were detected in non-rosy biofilms. The majority of sequences (70%) obtained from the bacterial communities of the three investigated rosy biofilms were related to sequences of the genus Rubrobacter (red-pigmented bacteria), demonstrating a correlation between Rubrobacter-related bacteria and the phenomenon of rosy discolouration of masonry and lime wall paintings.

Detection of Indigenous Halobacillus Populations in Damaged Ancient Wall Paintings and Building Materials: Molecular Monitoring and Cultivation

ABSTRACT Several moderately halophilic gram-positive, spore-forming bacteria have been isolated by conventional enrichment cultures from damaged medieval wall paintings and building materials. Enrichment and isolation were monitored by denaturing gradient gel electrophoresis and fluorescent in situ hybridization. 16S ribosomal DNA analysis showed that the bacteria are most closely related to Halobacillus litoralis. DNA-DNA reassociation experiments identified the isolates as a population of hitherto unknownHalobacillus species.

[29] Identification of archaea in objects of art by denaturing gradient gel electrophoresis analysis and shotgun cloning

Publisher Summary This chapter discusses the identification of archaea in objects of art by denaturing gradient gel electrophoresis analysis and shotgun cloning. Archaea are regarded as occurring in extreme habitats, such as hypersaline, anaerobic, alkaline, acidic, and hypenhermophilic environments. Further, they have not been previously regarded as important in global ecology. In this “extreme” environment, the formation of biofilms may enable archaea to survive exposure to suboptimal conditions. This chapter describes the development and application of a strategy combining denaturing gradient gel electrophoresis (DGGE) analysis and shotgun cloning for the analysis of archaea in biodeteriorated materials of cultural heritage. The combination of DGGE analysis with the construction of clone libraries to study archaeal communities has already been described here, but instead of DGGE, restriction fragment length polymorphism (RFLP) was used for the screening of clones. RFLP analysis of 16S rDNA has been used for several years as a method for comparing rDNAs. PCR products obtained with universal primers are digested with restriction enzymes. This method has been used for the study of communities, where the potentially large number of fragments can be resolved by using polyacrylamide gels to produce a communityspecific pattern. The combination of the methods described offers advantages over other methods that have been previously described: the parallel application of DGGE analysis and clone libraries combines the potential of both methods and overcomes their limitations; and the screening of the clones by DGGE analysis makes the procedure easier and faster than other molecular methods such as RFLP analysis.