VAAM-Jahrestagung 2011 Karlsruhe, 3.–6. April 2011

that can confer cell wall attachment, and a C-terminally located cysteine,histidine-dependent amidohydrolase/peptidase (CHAP) domain havingbacteriolytic activity in many proteins.Method: To characterize the functional domain structure of Aaa, wecontructed Aaa subclones expressing the N-terminal or C-terminal Aaadomainsin Escherichia coli and analyzed the functions of the respectivepurified proteins in various adherence assays and zymographic analysis.Results: We found that not only the bacteriolytic activity, but also adherenceto fibrinogen and fibronectin is mediated by the CHAP domain, thusdemonstrating for the first time an adhesive function for this domain. Incontrast, efficient adherence to endothelial cells and vitronectin requires thewhole Aaa. Adherence of an S. aureus aaa mutant and the complemented aaamutant is slightly decreased and increased, respectively, to vitronectin, butnot to fibrinogen and fibronectin, which might at least in part result from anincreased expression of the autolysin/adhesin Atl. Moreover, an S. aureus atlmutant showing enhanced adherence to extracellular matrix proteins andendothelial cells revealed increased aaa-expression and production of Aaa.Thus, the redundant functions of Aaa and Atl might at least in part beinterchangeable and furthermore be regulated by so far unknownmechanisms.Conclusion: In conclusion, the adhesive properties of Aaa might promote S.aureus colonization of host extracellular matrix and tissue and thus mightplay an important role in the pathogenesis of serious S. aureus infectionswith this pathogen.MPP032Antibiotic treatment provokes activity of IS256r inseveral S. aureus strainsM. Nagel, G. Bierbaum*Institute of Medical Microbiology, Immunology and Parasitology, UniversiyHospital, Bonn, GermanyMobile elements are wide-spread in nearly all bacterial species. After thefirst description of insertion sequences forty years ago, more than 500insertion sequences in 159 bacterial species have been described andcharacterised. Here we focus on IS256, a common element of staphylococci.IS elements have been shown to create mutations by insertion into andexcision from the genome, to confer genome plasticity and to conferresistance against antibiotics by insertion into promoter sequences or openreading frames.In order to test whether the presence of antibiotics leads to the mobilisationof IS elements in S. aureus, a system that measures the transpositionfrequency of IS256 was employed. This system comprised an IS256 elementthat had been tagged by an erythromycin marker (IS256r) and an inactivatedIS256 for control purposes [1].Treatment with subinhibitory concentrations of clinically relevant antibiotics(linezolid, ciprofloxacin and vancomycin) resulted in increases oftransposition frequency of IS256r which was highest in the presence ofciprofloxacin in S. aureus RN1-HG (restored rsbU). In conclusion, thereseems to be a correlation between antibiotic stress and mobilisation ofIS256. Interestingly, we observed that there is a higher transposition rate inSigmaB deficient strains like S. aureus 8325.The mechanism behind the activation of transposition is still poorlyunderstood. In order to elucidate this phenomenon, a putative SigmaBantisense promoter in the IS256r element was inactivated by site directedmutagenesis. The resulting clone showed an upregulation of transpositionactivity. Furthermore, the significance of a second putative antisensepromoter is still under investigation.([1] Valle, J. et al (2007): J. Bacteriol., 2886-2896.)MPP033Variation within a field population of Dickeyachrysanthemi in permissiveness for broad host-rangeplasmidsH. Heuer*, J. Ebers, N. Weinert, K. SmallaEpidemiology and Pathogen Diagnostics, Julius Kühn-Institut (JKI),Braunschweig, GermanyHorizontal gene transfer through broad host-range plasmids has the potentialto provide sufficient genetic flexibility to populations of Dickeyachrysanthemi to keep its phytopathogenic lifestyle efficient despite evolvingplant defences. However, foreign DNA often is deleterious for the individualcell. We investigated whether plasmid uptake varied among individualstrains of a field population to balance the benefit from genetic flexibilityand the cost on population-level. The transfer frequency of broad host-rangeIncP-1 plasmids between an Escherichia coli donor and Dickeyachrysanthemi strains significantly differed among isolates from a fieldpopulation. Transfer frequencies for two IncP-1 plasmids, pTH10 and pB10of the divergent a- and b-subgroups, respectively, correlated well. D.chrysanthemi strains, which differed in permissiveness for these plasmids byorders of magnitude, were indistinguishable by other phenotypic traits,genomic fingerprints, or by hrpN gene sequences. Such strains were isolatedin close vicinity. Spatial aggregation of subpopulations with increasedpermissiveness for plasmids was not observed, indicating a reasonably fastgenetic mechanism of switching in permissiveness. In contrast to IncP-1plasmids, transfer frequencies for the narrow host-range LowGC-typeplasmid pHHV216 were similar among strains suggesting that themechanism underlying the differential permissiveness did not target foreignDNA in general.MPP034Staphylococcal teichoic acidis regulate targeting of themajor autolysin Atl.M. Schlag*, R. Biswas, B. Krismer, F. GötzInstitute of Microbiology and Infection Medicine, Eberhard-Karls-University, Tübingen, GermanyStaphylococcal cell separation depends largely on the bifunctional autolysinAtl that is processed to amidase-R(1,2) and R(3)-glucosaminidase. Thesemurein hydrolases are targeted via repeat domains (R) to the septal region ofthe cell surface, thereby allowing localized peptidoglycan hydrolysis andseparation of the dividing cells. We could show that targeting of the amidaserepeats is based on an exclusion strategy mediated by wall teichoic acid(WTA). In Staphylococcus aureus wild-type, externally applied repeats(R(1,2)) or endogenously expressed amidase were localized exclusively atthe cross-wall region, while in ΔtagO mutant that lacks WTA autolysin wasevenly distributed on the cell surface, which explains the increased fragilityand autolysis susceptibility of the mutant. WTA prevented binding of Atl tothe old cell wall but not to the cross-wall region suggesting a lower WTAcontent. In binding studies with ConcanavalinA-fluorescein (ConA-FITC)conjugate that binds preferentially to teichoic acids, ConA-FITC was boundthroughout the cell surface with the exception of the cross wall. ConAbinding suggest that either content or polymerization of WTA graduallyincreases with distance from the cross-wall. By preventing binding of Atl,WTA directs Atl to the cross-wall to perform the last step of cell division,namely separation of the daughter cells.MPP035Comparative proteomics within the species Paenibacilluslarvae, a bacterial honey bee pathogenA. Fünfhaus*, E. GenerschState Institute for Bee Research, Department of Molecular Microbiologyand Bee Diseases, Hohen Neuendorf, GermanyRecently, four different genotypes (ERIC I - ERIC IV) of Paenibacilluslarvae, the causative agent of American Foulbrood (AFB) of honey bees,have been described [3]. The phenotypical differences between thesegenotypes included differences in metabolism [4], in colony and sporemorphology, and in virulence [2]. To identify factors (genes and proteins)putatively responsible especially for the observed differences in virulencewe applied comparative genomics via Suppression SubtractiveHybridization [1], 2009) and comparative proteomics via 2D-SDS-PAGEanalysis[5] followed by mass spectrometric identfication of differentiallyexpressed proteins. We here present our data on the successful developmentof (i) a protein extraction method for P. larvae suitable for subsequent 2D-SDS-PAGE analysis and (ii) reproducible 2D-SDS-PAGE-analyses of theseprotein preparations. Based on the obtained master protein patterns of thefour P. larvae -genotypes isolated from liquid bacterial cultures, weidentified several differentially expressed proteins presumably linked to theobserved phenotypic differences.[1] Fünfhaus, A. et al (2009): Use of suppression subtractive hybridization to identify geneticdifferences between differentially virulent genotypes of Paenibacillus larvae, the etiological agent ofAmerican foulbrood of honeybees. Environ. Microbiol. Reports 1, 240-250.[2] Genersch, E. et al (2005): Strain- and genotype-specific differences in virulence of Paenibacilluslarvae subsp. larvae, the causative agent of American foulbrood disease in honey bees. Appl. Environ.Microbiol. 71, 7551-7555.[3] Genersch, E. et al (2006): Reclassification of Paenibacillus larvae subsp. pulvifaciens andPaenibacillus larvae subsp. larvae as Paenibacillus larvae without subspecies differentiation. Int. J.Syst. Evol. Microbiol. 56, 501-511.spektrum | Tagungsband 2011