Comprehensive transposon mutant library of Pseudomonas aeruginosa | PNAS 3, 109136 ( 1996). GMS Hyg. P. aeruginosa appears to have the most complex chemosensory systems of all the complete bacterial genomes, with four loci that encode probable chemotaxis signal-transduction pathways (see Supplementary Information ). Mutational events were already reported in the 1970s (10), and more recently sequence variations of MexT, which regulates the MexEF-OprN multidrug efflux system, were described (18, 24). P. aeruginosa is a versatile Gram-negative bacterium that grows in soil, marshes and coastal marine habitats, as well as on plant and animal tissues1. Sign up for the Nature Briefing newsletter what matters in science, free to your inbox daily. This microorganism is an important laboratory model and a mainstay for. There are nearly 50% more paralogous groups in P.aeruginosa than predicted on the basis of a simple comparison of genome sizes. Strain PAO1, a wound isolate36, was chosen as a strain prototype for sequencing because it is the most widely used P. aeruginosa laboratory strain and because physical and genetic maps were available12,13. An unexpected finding was that many of the xcp genes have homologues elsewhere on the chromosome, including four additional homologues of xcpQ and xcpR. The efficacy of biosynthesized silver nanoparticles against Pseudomonas aeruginosa isolates from cystic fibrosis patients, Cross-talk between cancer and Pseudomonas aeruginosa mediates tumor suppression, The impact of biosynthesized ZnO nanoparticles from Olea europaea (Common Olive) on Pseudomonas aeruginosa growth and biofilm formation, Quorum sensing activities and genomic insights of plant growth-promoting rhizobacteria isolated from Assam tea, Integrated metabolomic and transcriptomic analyses of the synergistic effect of polymyxinrifampicin combination against Pseudomonas aeruginosa, Cancel 31 , 394395 (1999). Bethesda, MD 20894, Web Policies KEGG GENOME: Pseudomonas aeruginosa PAO1 Kunst, F. et al. Tseng, T.-T. et al. The sequence reads were mapped to the PAO1-UW reference sequence ({"type":"entrez-nucleotide","attrs":{"text":"NC_002516","term_id":"110645304","term_text":"NC_002516"}}NC_002516) using Illumina's ELAND aligner (efficient large-scale alignment of nucleotide databases). Published ahead of print on 18 December 2009. USA 86, 424428 (1989). document the treatment, and clinical improvement, of a male patient with a life-threatening chronic multi-drug resistant Pseudomonas aeruginosa infection with aerosolized . Pseudomonas aeruginosa. Sanderson ND, Kapel N, Rodger G, Webster H, Lipworth S, Street TL, Peto T, Crook D, Stoesser N. Microb Genom. was recipient of the Medical Research Council of Canada (MRC) Distinguished Scientist Award and received funding from the MRC. P. aeruginosa secretes several virulence factors, including toxins, lipases and proteases. Pseudomonas aeruginosa Genomic Structure and Diversity 11, 137 153 (1994). Enhanced annotations and features for comparing thousands of Pseudomonas genomes in the Pseudomonas genome database. Reyne N, McCarron A, Cmielewski P, Parsons D, Donnelley M. Front Physiol. The exception is the inversion of more than one-quarter of the genome in the PAO1 isolate we sequenced, relative to DSM-1707, the PAO1-derived isolate previously mapped in the laboratory of B. Tmmler12,13 (Fig. While the two strains have contributed to a wide breadth of knowledge on the natural behaviors and therapeutic susceptibilities of P. aeruginosa, they have demonstrated significant deviations from observations in human infections. The part of the genome shown in gray is inverted in PAO1-UW in contrast to PAO1-DSM and other sublines. Microbiol. Pseudomonas aeruginosa PAO1 is the most commonly used strain for research on this ubiquitous and metabolically versatile opportunistic pathogen. Closer inspection of the sequenced DNA sample of the PAO1-DSM strain, however, revealed a G-T transversion in 9 of 79 sequence reads, resulting in a change of arginine to leucine in about 10% of the population. Control. Thank you! Mice were sacrificed after 48 h for the evaluation of lung histology or the determination of CFU in homogenized organs (lungs, liver, spleen, and brain). Dis Markers. The Pseudomonas Genome Database collaborates with an Over decades discordant phenotypes of PAO1 sublines have emerged. Comparison of the genome sequence with the physical map revealed a large, 2.2-Mb inversion between the sequenced PAO1-UW strain (36) and the original PAO1 strain (9, 10), indicating that PAO1 sublines maintained worldwide in numerous laboratories and strain collections had diversified their genomic sequence. Genomic differences in PAO1 sublines. In addition to known oxidative enzymes and pathways, we found a substantial number of other genes encoding putative enzymes characteristic of -oxidation, such as acyl-CoA dehydrogenase (25 genes) and enoyl-CoA hydratase/isomerase (16 genes). Fischer S, Dethlefsen S, Klockgether J, Tmmler B. mSystems. Would you like email updates of new search results? Federal government websites often end in .gov or .mil. Future Microbiol. In each case the literature was searched to ensure that the proteins encoded by the homologous genes were functionally characterized to avoid the perpetuation of poorly supported functional assignments. (Top row) Infection with 106 CFU of all three PAO1 strains has induced a moderate acute fibrinogenic pneumonia, mostly confined to the alveoli. Figure Figure22 shows the genome map positions of the SNPs. Moulton, R. C. & Montie, R. C. Chemotaxis by Pseudomonas aeruginosa. An aliquot was transferred into 20 ml LB adjusted to an optical density at 578 nm (OD578) of 0.1 and cultivated for about 6 h until an OD578 of 2.5 was reached. Comparative analysis of digests of PAO1 and DSM-1707 with SfoI, SwaI and PacI supported this possibility. Mucinophilic and chemotactic properties of Pseudomonas aeruginosa in relation to pulmonary colonization in cystic fibrosis. Front. Nucleic Acids Res. Schmidt, K. D., Schmidt-Rose, T., Rmling, U. Kato, J., Sakai, Y., Nikata, T. & Ohtake, H. Cloning and characterization of a Pseudomonas aeruginosa gene involved in the negative regulation of phosphate taxis. Open reading frames were initially predicted by GeneMark.HMM (http://genemark.biology.gatech.edu/GeneMark/whmm.cgi)39. & Filloux, A. Structure-function analysis of XcpP, a component involved in general secretory pathway-dependent protein secretion in Pseudomonas aeruginosa. PseudoCAP was the first wholly Internet-based and First, the PAO1-UW subline maintained in our laboratory showed intragenic deletions in PA1695 and PA3760 compared to the published PAO1-UW sequence. The variable presence of RGP42 in PAO1-UW and PAO1-UW is consistent with our notion during physical mapping that the diagnostic SpeI fragment exhibited a variable intensity of the ethidium bromide stain in separate PFGE runs, implying that this prophage region is prone to deletion. Each of the two remaining clusters has homology tothe che genes from E. coli and to the frz genes from the non-flagellated gliding bacterium, Myxococcus xanthus29. Unable to load your collection due to an error, Unable to load your delegates due to an error. Physical mapping and whole-genome resequencing of PAO1 sublines uncovered an extended genomic diversity between the two strains that were the basis for the initial sequencing project and the first genome-wide mutant library. Thus, a Qconsensus (s) value of 1 indicates identical base calls for position s in all covering reads, while lower values are caused by heterogeneous results. DSM 1707). Consistent with its increased genetic complexity, P.aeruginosa has the greatest percentage of genes devoted to command and control systems (for example, environmental sensors and transcriptional regulators) observed in a bacterial genome. Overview: bqsR, Pseudomonas aeruginosa PAO1 - Pseudomonas genome database The PAO1 sublines differed in their ability to cope with nutrient limitation and their virulence in an acute murine airway infection model. Curr Opin Pulm Med. JBrowse: Annotated Features. With the advent of pulsed-field gel electrophoresis (PFGE), a physical map of the PAO1 genome was constructed (32) and later merged with the genetic map information (12). (2016) doi: 10.1093/nar/gkv1227 (Database issue). Open Access Published: 08 October 2021 An updated genome-scale metabolic network reconstruction of Pseudomonas aeruginosa PA14 to characterize mucin-driven shifts in bacterial metabolism. 28, 7780 (2000). These infections are impossible to eradicate, in part because of the natural resistance of the bacterium to antibiotics, and ultimately lead to pulmonary failure and death. Funding for this work is gratefully provided by Cystic Fibrosis The threshold for a reliable SNP in the primary sequence data was set to be coverage of at least 10-fold and a Qconsensus of 0.9 or more (see above). Further, in addition to the genes known to be involved in synthesis of lipopolysaccharides, we noted three other genetic loci that may be involved in the synthesis of extracellular polysaccharides (see Supplementary Information). The PAO1 genome appears to contain four additional type I systems. (Fig.2).2). DO is the President and CEO of Owen Biosciences Inc. & Goffaux, F. Secretion of virulence factors by Escherichia coli. Lett. Thus, no correlation was observed between divergent susceptibilities to antimicrobials in PAO1 sublines and the detected SNPs in loci mexT and fusA1 that are known to confer natural antibiotic resistance. (Fig.1D).1D). 2023 Jan;9(1):mgen000910. Here we report the complete sequence of P. aeruginosa strain PAO1. USA 95, 1389913904 (1998). CAS official website and that any information you provide is encrypted 58, 14891495 (1990). 1). Overview: pilO, Pseudomonas aeruginosa PAO1 Nelson, J. W. et al. Restriction fragments that are altered due to the chromosomal inversion are labeled. The six different culturing conditions are described in Table S2 in the supplemental material. Thirty-six cycles of sequencing-by-synthesis were performed for each library in four lanes of a flow cell with the Genome Analyzer I. Illumina Genome Analyzer Pipeline version 0.2 software was used to qualify reads passing default signal quality filters. For the last three organisms, these numbers are based on predictions taken from http://www-biology.ucsd.edu/ipaulsen/transport/. P.aeruginosa potentially possesses four chemotaxis systems, at least one of which contributes to its ability to form biofilms35. Please note that the ter locus is located in PAO1 sublines PAO1-DSM and MPAO1 opposite the origin of replication (Fig. Microbiol. A major factor in its prominence as a pathogen is its. Article Strain PAO1, a derivative of the original Australian PAO isolate, has been distributed worldwide to laboratories and strain collections. & Rmling, U. Clipboard, Search History, and several other advanced features are temporarily unavailable. The inversion in PAO1-UW must have occurred after the PAO1 strain had been deposited by the original investigator in public strain collections. Toward the end of August 2000, the 6.3 Mbp whole genome sequence of Pseudomonas aeruginosa strain PAO1 was published. Top Mizzou Basketball Players, Burial Beer Charlotte, The Farm A Gathering Place Wedding Cost, Fm San Luis Obispo Radio Stations, Elathottam For Sale Near Hamburg, Articles P
" />

pseudomonas aeruginosa pao1 genome

pseudomonas aeruginosa pao1 genome

2023 Mar 13;14:1095928. doi: 10.3389/fmicb.2023.1095928. Dis. In conclusion, P. aeruginosa PAO1 shows an ongoing microevolution of genotype and phenotype that jeopardizes the reproducibility of research. 25, 33893402 (1997). A genetic map of its chromosome was generated by exploiting the mechanisms of gene exchange in bacteria, i.e., transduction and conjugation (11). Get the most important science stories of the day, free in your inbox. international panel of expert Pseudomonas researchers to 137, 274280 (1979). To verify the correct dilution, an aliquot was serially diluted on LB agar plates. HHS Vulnerability Disclosure, Help An instructive example within our project is the known hot spot of mutation in the P. aeruginosa genome, the gene lasR. Today 24, 350359. In contrast to its limited ability to grow on sugars, P. aeruginosa can use a wide variety of other carbon compounds, and its genome provides insight into the molecular basis of this metabolic versatility. (Manual annotation of the genome identified 521 genes (9.4%) as encoding either transcriptional regulators or two-component regulatory system proteins (Table 2). In contrast, P. aeruginosa has only 3040% of the number of predicted genes present in the simple metazoans Caenorhabditis elegans and Drosophila melanogaster6. If you have used this database, please ensure that you acknowledge this most recent Pseudomonas Genome Database publication rather than just the website URL. the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in HHS Vulnerability Disclosure, Help An official website of the United States government. 27, S93S99 (1998). Many of these deviations are related to experimental inconsistencies in laboratory strain environment that complicate and, at times, terminate translation from laboratory results to clinical applications. Genomic and Phenotypic Diversity among Ten Laboratory Isolates of. sharing sensitive information, make sure youre on a federal Nucleic Acids Res. Pathohistological findings in C3H/HeN murine lungs 48 h after intratracheal, MeSH Database now has 7960 P. aeruginosa genomes. Microbiol. Vet. Print 2019 Nov 1. Database development is supported by key funding from: Pseudomonas Genome Database version 21.1 released. With 5570 open reading frames (ORFs), PAO1 had the largest microbial genome sequenced up to that point in timeincluding a large proportion of metabolic, transport and antimicrobial resistance genes supporting its ability to colonize diverse environments. The genomes analysed were M. genitalium (480 predicted ORFs), M. pneumoniae (677), R. prowazekii (834), B. burgdorferi (850), C. trachomatis (894), T. pallidum (1031), C. pneumoniae (1052), A. aeolicus (1522), H. pylori 26695 (1553), H. influenzae (1709), M. jannaschii (1715), P. abyssi (1765), T. maritima (1846), M. thermoautotrophicum (1869), N. meningitidis MC58 (2025), P. horikoshii (2064), A.fulgidus (2407), Synechocystis PCC6803 (3169), M. tuberculosis (3918), B. subtilis (4100), E. coli (4289), P. aeruginosa (5570). Dis. The metabolically versatile Pseudomonas aeruginosa is an opportunistic pathogen of plants, animals, and humans and is ubiquitously distributed in soil and aquatic habitats. -, Azimi S., Kafil H. S., Baghi H. B., Shokrian S., Najaf K., Asgharzadeh M., et al. (Fig.1A;1A; see also Table S3 and Fig. doi: 10.1128/mSystems.01007-20. Gordon, D., Abajian, C. & Green, P. Consed: a graphical tool for sequence finishing. Careers. Generally, the acquisition of genes in the form of pathogenicity islands distinguishes pathogenic isolates from nonpathogens. Comprehensive transposon mutant library of Pseudomonas aeruginosa | PNAS 3, 109136 ( 1996). GMS Hyg. P. aeruginosa appears to have the most complex chemosensory systems of all the complete bacterial genomes, with four loci that encode probable chemotaxis signal-transduction pathways (see Supplementary Information ). Mutational events were already reported in the 1970s (10), and more recently sequence variations of MexT, which regulates the MexEF-OprN multidrug efflux system, were described (18, 24). P. aeruginosa is a versatile Gram-negative bacterium that grows in soil, marshes and coastal marine habitats, as well as on plant and animal tissues1. Sign up for the Nature Briefing newsletter what matters in science, free to your inbox daily. This microorganism is an important laboratory model and a mainstay for. There are nearly 50% more paralogous groups in P.aeruginosa than predicted on the basis of a simple comparison of genome sizes. Strain PAO1, a wound isolate36, was chosen as a strain prototype for sequencing because it is the most widely used P. aeruginosa laboratory strain and because physical and genetic maps were available12,13. An unexpected finding was that many of the xcp genes have homologues elsewhere on the chromosome, including four additional homologues of xcpQ and xcpR. The efficacy of biosynthesized silver nanoparticles against Pseudomonas aeruginosa isolates from cystic fibrosis patients, Cross-talk between cancer and Pseudomonas aeruginosa mediates tumor suppression, The impact of biosynthesized ZnO nanoparticles from Olea europaea (Common Olive) on Pseudomonas aeruginosa growth and biofilm formation, Quorum sensing activities and genomic insights of plant growth-promoting rhizobacteria isolated from Assam tea, Integrated metabolomic and transcriptomic analyses of the synergistic effect of polymyxinrifampicin combination against Pseudomonas aeruginosa, Cancel 31 , 394395 (1999). Bethesda, MD 20894, Web Policies KEGG GENOME: Pseudomonas aeruginosa PAO1 Kunst, F. et al. Tseng, T.-T. et al. The sequence reads were mapped to the PAO1-UW reference sequence ({"type":"entrez-nucleotide","attrs":{"text":"NC_002516","term_id":"110645304","term_text":"NC_002516"}}NC_002516) using Illumina's ELAND aligner (efficient large-scale alignment of nucleotide databases). Published ahead of print on 18 December 2009. USA 86, 424428 (1989). document the treatment, and clinical improvement, of a male patient with a life-threatening chronic multi-drug resistant Pseudomonas aeruginosa infection with aerosolized . Pseudomonas aeruginosa. Sanderson ND, Kapel N, Rodger G, Webster H, Lipworth S, Street TL, Peto T, Crook D, Stoesser N. Microb Genom. was recipient of the Medical Research Council of Canada (MRC) Distinguished Scientist Award and received funding from the MRC. P. aeruginosa secretes several virulence factors, including toxins, lipases and proteases. Pseudomonas aeruginosa Genomic Structure and Diversity 11, 137 153 (1994). Enhanced annotations and features for comparing thousands of Pseudomonas genomes in the Pseudomonas genome database. Reyne N, McCarron A, Cmielewski P, Parsons D, Donnelley M. Front Physiol. The exception is the inversion of more than one-quarter of the genome in the PAO1 isolate we sequenced, relative to DSM-1707, the PAO1-derived isolate previously mapped in the laboratory of B. Tmmler12,13 (Fig. While the two strains have contributed to a wide breadth of knowledge on the natural behaviors and therapeutic susceptibilities of P. aeruginosa, they have demonstrated significant deviations from observations in human infections. The part of the genome shown in gray is inverted in PAO1-UW in contrast to PAO1-DSM and other sublines. Microbiol. Pseudomonas aeruginosa PAO1 is the most commonly used strain for research on this ubiquitous and metabolically versatile opportunistic pathogen. Closer inspection of the sequenced DNA sample of the PAO1-DSM strain, however, revealed a G-T transversion in 9 of 79 sequence reads, resulting in a change of arginine to leucine in about 10% of the population. Control. Thank you! Mice were sacrificed after 48 h for the evaluation of lung histology or the determination of CFU in homogenized organs (lungs, liver, spleen, and brain). Dis Markers. The Pseudomonas Genome Database collaborates with an Over decades discordant phenotypes of PAO1 sublines have emerged. Comparison of the genome sequence with the physical map revealed a large, 2.2-Mb inversion between the sequenced PAO1-UW strain (36) and the original PAO1 strain (9, 10), indicating that PAO1 sublines maintained worldwide in numerous laboratories and strain collections had diversified their genomic sequence. Genomic differences in PAO1 sublines. In addition to known oxidative enzymes and pathways, we found a substantial number of other genes encoding putative enzymes characteristic of -oxidation, such as acyl-CoA dehydrogenase (25 genes) and enoyl-CoA hydratase/isomerase (16 genes). Fischer S, Dethlefsen S, Klockgether J, Tmmler B. mSystems. Would you like email updates of new search results? Federal government websites often end in .gov or .mil. Future Microbiol. In each case the literature was searched to ensure that the proteins encoded by the homologous genes were functionally characterized to avoid the perpetuation of poorly supported functional assignments. (Top row) Infection with 106 CFU of all three PAO1 strains has induced a moderate acute fibrinogenic pneumonia, mostly confined to the alveoli. Figure Figure22 shows the genome map positions of the SNPs. Moulton, R. C. & Montie, R. C. Chemotaxis by Pseudomonas aeruginosa. An aliquot was transferred into 20 ml LB adjusted to an optical density at 578 nm (OD578) of 0.1 and cultivated for about 6 h until an OD578 of 2.5 was reached. Comparative analysis of digests of PAO1 and DSM-1707 with SfoI, SwaI and PacI supported this possibility. Mucinophilic and chemotactic properties of Pseudomonas aeruginosa in relation to pulmonary colonization in cystic fibrosis. Front. Nucleic Acids Res. Schmidt, K. D., Schmidt-Rose, T., Rmling, U. Kato, J., Sakai, Y., Nikata, T. & Ohtake, H. Cloning and characterization of a Pseudomonas aeruginosa gene involved in the negative regulation of phosphate taxis. Open reading frames were initially predicted by GeneMark.HMM (http://genemark.biology.gatech.edu/GeneMark/whmm.cgi)39. & Filloux, A. Structure-function analysis of XcpP, a component involved in general secretory pathway-dependent protein secretion in Pseudomonas aeruginosa. PseudoCAP was the first wholly Internet-based and First, the PAO1-UW subline maintained in our laboratory showed intragenic deletions in PA1695 and PA3760 compared to the published PAO1-UW sequence. The variable presence of RGP42 in PAO1-UW and PAO1-UW is consistent with our notion during physical mapping that the diagnostic SpeI fragment exhibited a variable intensity of the ethidium bromide stain in separate PFGE runs, implying that this prophage region is prone to deletion. Each of the two remaining clusters has homology tothe che genes from E. coli and to the frz genes from the non-flagellated gliding bacterium, Myxococcus xanthus29. Unable to load your collection due to an error, Unable to load your delegates due to an error. Physical mapping and whole-genome resequencing of PAO1 sublines uncovered an extended genomic diversity between the two strains that were the basis for the initial sequencing project and the first genome-wide mutant library. Thus, a Qconsensus (s) value of 1 indicates identical base calls for position s in all covering reads, while lower values are caused by heterogeneous results. DSM 1707). Consistent with its increased genetic complexity, P.aeruginosa has the greatest percentage of genes devoted to command and control systems (for example, environmental sensors and transcriptional regulators) observed in a bacterial genome. Overview: bqsR, Pseudomonas aeruginosa PAO1 - Pseudomonas genome database The PAO1 sublines differed in their ability to cope with nutrient limitation and their virulence in an acute murine airway infection model. Curr Opin Pulm Med. JBrowse: Annotated Features. With the advent of pulsed-field gel electrophoresis (PFGE), a physical map of the PAO1 genome was constructed (32) and later merged with the genetic map information (12). (2016) doi: 10.1093/nar/gkv1227 (Database issue). Open Access Published: 08 October 2021 An updated genome-scale metabolic network reconstruction of Pseudomonas aeruginosa PA14 to characterize mucin-driven shifts in bacterial metabolism. 28, 7780 (2000). These infections are impossible to eradicate, in part because of the natural resistance of the bacterium to antibiotics, and ultimately lead to pulmonary failure and death. Funding for this work is gratefully provided by Cystic Fibrosis The threshold for a reliable SNP in the primary sequence data was set to be coverage of at least 10-fold and a Qconsensus of 0.9 or more (see above). Further, in addition to the genes known to be involved in synthesis of lipopolysaccharides, we noted three other genetic loci that may be involved in the synthesis of extracellular polysaccharides (see Supplementary Information). The PAO1 genome appears to contain four additional type I systems. (Fig.2).2). DO is the President and CEO of Owen Biosciences Inc. & Goffaux, F. Secretion of virulence factors by Escherichia coli. Lett. Thus, no correlation was observed between divergent susceptibilities to antimicrobials in PAO1 sublines and the detected SNPs in loci mexT and fusA1 that are known to confer natural antibiotic resistance. (Fig.1D).1D). 2023 Jan;9(1):mgen000910. Here we report the complete sequence of P. aeruginosa strain PAO1. USA 95, 1389913904 (1998). CAS official website and that any information you provide is encrypted 58, 14891495 (1990). 1). Overview: pilO, Pseudomonas aeruginosa PAO1 Nelson, J. W. et al. Restriction fragments that are altered due to the chromosomal inversion are labeled. The six different culturing conditions are described in Table S2 in the supplemental material. Thirty-six cycles of sequencing-by-synthesis were performed for each library in four lanes of a flow cell with the Genome Analyzer I. Illumina Genome Analyzer Pipeline version 0.2 software was used to qualify reads passing default signal quality filters. For the last three organisms, these numbers are based on predictions taken from http://www-biology.ucsd.edu/ipaulsen/transport/. P.aeruginosa potentially possesses four chemotaxis systems, at least one of which contributes to its ability to form biofilms35. Please note that the ter locus is located in PAO1 sublines PAO1-DSM and MPAO1 opposite the origin of replication (Fig. Microbiol. A major factor in its prominence as a pathogen is its. Article Strain PAO1, a derivative of the original Australian PAO isolate, has been distributed worldwide to laboratories and strain collections. & Rmling, U. Clipboard, Search History, and several other advanced features are temporarily unavailable. The inversion in PAO1-UW must have occurred after the PAO1 strain had been deposited by the original investigator in public strain collections. Toward the end of August 2000, the 6.3 Mbp whole genome sequence of Pseudomonas aeruginosa strain PAO1 was published.

Top Mizzou Basketball Players, Burial Beer Charlotte, The Farm A Gathering Place Wedding Cost, Fm San Luis Obispo Radio Stations, Elathottam For Sale Near Hamburg, Articles P

%d bloggers like this: