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High-Throughput Method for Rapid Induction of Prophages from Lysogens and Its Application in the Study of Shiga Toxin-Encoding Escherichia coli Strains ▿
McDonald, James E.
Smith, Darren L.
Fogg, Paul C. M.
McCarthy, Alan J.
Allison, Heather E.
Applied and Environmental Microbiology
A high-throughput 96-well plate-based method for the rapid induction of endogenous prophages from individual bacterial strains was developed. The detection of endogenous prophages was achieved by the filtration of the culture liquor following norfloxacin induction and subsequent PCRs targeting bacteriophage-carried gene markers. The induction method was tested on 188 putative Shiga toxin (Stx)-producing Escherichia coli (STEC) strains and demonstrated the ability to detect both lambdoid and stx-carrying bacteriophages in strains for which plaques were not observed via plaque assay. Lambdoid bacteriophages were detected in 37% of the induced phage preparations via amplification of the Q gene, and Stx1- and Stx2-encoding phages were detected in 2 and 14% of the strains, respectively. The method therefore provided greater sensitivity for the detection of Stx and other lambdoid bacteriophage populations carried by STEC strains than that for the established method of plaque assay using bacterial indicator strains, enabling, for the first time, large-scale bacteriophage population and diversity studies.
Related Articles
Different Expression Patterns of Genes from the Exo-Xis Region of Bacteriophage λ and Shiga Toxin-Converting Bacteriophage Ф24B following Infection or Prophage Induction in Escherichia coli
Bloch, Sylwia
Nejman-Faleńczyk, Bożena
Dydecka, Aleksandra
Łoś, Joanna M.
Felczykowska, Agnieszka
Węgrzyn, Alicja
Węgrzyn, Grzegorz
PLoS ONE
Lambdoid bacteriophages serve as useful models in microbiological and molecular studies on basic biological process. Moreover, this family of viruses plays an important role in pathogenesis of enterohemorrhagic Escherichia coli (EHEC) strains, as they are carriers of genes coding for Shiga toxins. Efficient expression of these genes requires lambdoid prophage induction and multiplication of the phage genome. Therefore, understanding the mechanisms regulating these processes appears essential for both basic knowledge and potential anti-EHEC applications. The exo-xis region, present in genomes of lambdoid bacteriophages, contains highly conserved genes of largely unknown functions. Recent report indicated that the Ea8.5 protein, encoded in this region, contains a newly discovered fused homeodomain/zinc-finger fold, suggesting its plausible regulatory role. Moreover, subsequent studies demonstrated that overexpression of the exo-xis region from a multicopy plasmid resulted in impaired lysogenization of E. coli and more effective induction of λ and Ф24B prophages. In this report, we demonstrate that after prophage induction, the increase in phage DNA content in the host cells is more efficient in E. coli bearing additional copies of the exo-xis region, while survival rate of such bacteria is lower, which corroborated previous observations. Importantly, by using quantitative real-time reverse transcription PCR, we have determined patterns of expressions of particular genes from this region. Unexpectedly, in both phages λ and Ф24B, these patterns were significantly different not only between conditions of the host cells infection by bacteriophages and prophage induction, but also between induction of prophages with various agents (mitomycin C and hydrogen peroxide). This may shed a new light on our understanding of regulation of lambdoid phage development, depending on the mode of lytic cycle initiation.
Related Articles
Stability and Infectivity of Cytolethal Distending Toxin Type V Gene-Carrying Bacteriophages in a Water Mesocosm and under Different Inactivation Conditions
Allué-Guardia, Anna
Jofre, Juan
Muniesa, Maite
Applied and Environmental Microbiology
Two cytolethal distending toxin (Cdt) type V-encoding bacteriophages (Φ62 and Φ125) were induced spontaneously from their wild-type Escherichia coli strains and from the lysogens generated in Shigella sonnei. The stability of Cdt phages was determined at various temperatures and pH values after 1 month of storage by means of infectivity tests using a plaque blot assay and analysis of phage genomes using real-time quantitative PCR (qPCR): both were highly stable. We assessed the inactivation of Cdt phages by thermal treatment, chlorination, UV radiation, and in a mesocosm in both summer and winter. The results for the two Cdt phages showed similar trends and were also similar to the phage SOM23 used for reference, but they showed a much higher persistence than Cdt-producing E. coli. Cdt phages showed maximal inactivation after 1 h at 70°C, 30 min of UV radiation, and 30 min of contact with a 10-ppm chlorine treatment. Inactivation in a mesocosm was higher in summer than in winter, probably because of solar radiation. The treatments reduced the number of infectious phages but did not have a significant effect on the Cdt phage particles detected by qPCR. Cdt phages were quantified by qPCR in 73% of river samples, and these results suggest that Cdt phages are a genetic vehicle and the natural reservoir for cdt in the environment.
Related Articles
High Temperature in Combination with UV Irradiation Enhances Horizontal Transfer of stx2 Gene from E. coli O157:H7 to Non-Pathogenic E. coli
Yue, Wan-Fu
Du, Min
Zhu, Mei-Jun
PLoS ONE
Shiga toxin (stx) genes have been transferred to numerous bacteria, one of which is E. coli O157:H7. It is a common belief that stx gene is transferred by bacteriophages, because stx genes are located on lambdoid prophages in the E. coli O157:H7 genome. Both E. coli O157:H7 and non-pathogenic E. coli are highly enriched in cattle feedlots. We hypothesized that strong UV radiation in combination with high temperature accelerates stx gene transfer into non-pathogenic E. coli in feedlots.
Methodology/Principal Findings
E. coli O157:H7 EDL933 strain were subjected to different UV irradiation (0 or 0.5 kJ/m2) combination with different temperature (22, 28, 30, 32, and 37°C) treatments, and the activation of lambdoid prophages was analyzed by plaque forming unit while induction of Stx2 prophages was quantified by quantitative real-time PCR. Data showed that lambdoid prophages in E. coli O157:H7, including phages carrying stx2, were activated under UV radiation, a process enhanced by elevated temperature. Consistently, western blotting analysis indicated that the production of Shiga toxin 2 was also dramatically increased by UV irradiation and high temperature. In situ colony hybridization screening indicated that these activated Stx2 prophages were capable of converting laboratory strain of E. coli K12 into new Shiga toxigenic E. coli, which were further confirmed by PCR and ELISA analysis.
These data implicate that high environmental temperature in combination with UV irradiation accelerates the spread of stx genes through enhancing Stx prophage induction and Stx phage mediated gene transfer. Cattle feedlot sludge are teemed with E. coli O157:H7 and non-pathogenic E. coli, and is frequently exposed to UV radiation via sunlight, which may contribute to the rapid spread of stx gene to non-pathogenic E. coli and diversity of shiga toxin producing E. coli.
Related Articles
Shiga Toxin 2-Encoding Bacteriophages in Human Fecal Samples from Healthy Individuals
Martinez-Castillo, Alexandre
Quirós, Pablo
Navarro, Ferran
Miró, Elisenda
Muniesa, Maite
Applied and Environmental Microbiology
Shiga toxin-converting bacteriophages (Stx phages) carry the stx gene and convert nonpathogenic bacterial strains into Shiga toxin-producing bacteria. Previous studies have shown that high densities of free and infectious Stx phages are found in environments polluted with feces and also in food samples. Taken together, these two findings suggest that Stx phages could be excreted through feces, but this has not been tested to date. In this study, we purified Stx phages from 100 fecal samples from 100 healthy individuals showing no enteric symptoms. The phages retrieved from each sample were then quantified by quantitative PCR (qPCR). In total, 62% of the samples carried Stx phages, with an average value of 2.6 × 104 Stx phages/g. This result confirms the excretion of free Stx phages by healthy humans. Moreover, the Stx phages from feces were able to propagate in enrichment cultures of stx-negative Escherichia coli (strains C600 and O157:H7) and in Shigella sonnei, indicating that at least a fraction of the Stx phages present were infective. Plaque blot hybridization revealed lysis by Stx phages from feces. Our results confirm the presence of infectious free Stx phages in feces from healthy persons, possibly explaining the environmental prevalence observed in previous studies. It cannot be ruled out, therefore, that some positive stx results obtained during the molecular diagnosis of Shiga toxin-producing Escherichia coli (STEC)-related diseases using stool samples are due to the presence of Stx phages.
Related Articles
Isolation and genomic characterization of SfI, a serotype-converting bacteriophage of Shigella flexneri
Sun, Qiangzheng
Lan, Ruiting
Wang, Yiting
Wang, Jianping
Wang, Yan
Li, Peijing
Du, Pengcheng
Xu, Jianguo
BMC Microbiology
All Shigella flexneri serotypes except serotype 6 share a common O-antigen tetrasaccharide backbone and nearly all variations between serotypes are due to glucosyl and/or O-acetyl modifications of the common O unit mediated by glycosyltransferases encoded by serotype-converting bacteriophages. Several S. flexneri serotype-converting phages including SfV, SfX, Sf6 and SfII have been isolated and characterized. However, S. flexneri serotype-converting phage SfI which encodes a type I modification of serotype 1 (1a, 1b, 1c and 1d) had not yet been characterized.
The SfI phage was induced and purified from a S. flexneri serotype 1a clinical strain 019. Electron microscopy showed that the SfI phage has a hexagonal head and a long contractile tail, characteristic of the members of Myoviridae family. SfI can convert serotype Y to serotype 1a and serotype X to serotype 1d, but cannot convert 10 other S. flexneri serotypes (1a, 1b, 2a, 2b, 3a, 3b, 4a, 4b, 5a, Xv) tested, suggesting that SfI has a narrow host range. Similar to other S. flexneri serotype-converting phages, SfI integrates into the tRNA-thrW gene adjacent to proA of the host chromosome when lysogenized. The complete sequence of the SfI genome was 38,389 bp, encoding 66 open reading frames and two tRNA genes. Phage SfI shares significant homology with S. flexneri phage SfV, Escherichia coli prophage e14 and lambda, and is classified into the lambdoid phage family. SfI was found to use a cos mechanism for DNA packaging similar to that of phage SfV.
SfI contains features of lambdoid phages and is closely related to S. flexneri phage SfV, E. coli prophage e14 and lambda. The characterization of SfI enhances our understanding of serotype conversion of S. flexneri.
Related Articles
Characterization of the cryptic lambdoid prophage DLP12 of Escherichia coli and overlap of the DLP12 integrase gene with the tRNA gene argU.
Lindsey, D F
Mullin, D A
Walker, J R
Journal of Bacteriology
The argU (dnaY) gene of Escherichia coli is located, in clockwise orientation, at 577.5 kilobases (kb) on the chromosome physical map. There was a cryptic prophage spanning the 2 kb immediately downstream of argU that consisted of sequences similar to the phage P22 int gene, a portion of the P22 xis gene, and portions of the exo, P, and ren genes of bacteriophage lambda. This cryptic prophage was designated DLP12, for defective lambdoid prophage at 12 min. Immediately clockwise of DLP12 was the IS3 alpha 4 beta 4 insertion element. The argU and DLP12 int genes overlapped at their 3' ends, and argU contained sequence homologous to a portion of the phage P22 attP site. Additional homologies to lambdoid phages were found in the 25 kb clockwise of argU. These included the cryptic prophage qsr' (P. J. Highton, Y. Chang, W. R. Marcotte, Jr., and C. A. Schnaitman, J. Bacteriol. 162:256-262, 1985), a sequence homologous to a portion of lambda orf-194, and an attR homolog. Inasmuch as the DLP12 att int xis exo P/ren region, the qsr' region, and homologs of orf-194 and attR were arranged in the same order and orientation as the lambdoid prophage counterparts, we propose that the designation DLP12 be applied to all these sequences. This organization of the DLP12 sequences and the presence of the argU/DLP12 int pair in several E. coli strains and closely related species suggest that DLP12 might be an ancestral lambdoid prophage. Moreover, the presence of similar sequences at the junctions of DLP12 segments and their phage counterparts suggests that a common mechanism could have transferred these DLP12 segments to more recent phages.
Related Articles
Multilocus Characterization Scheme for Shiga Toxin-Encoding Bacteriophages▿
Smith, Darren L.
Wareing, Brian M.
Fogg, Paul C. M.
Riley, Laura M.
Spencer, Matthew
Cox, Michael J.
Saunders, Jon R.
McCarthy, Alan J.
Allison, Heather E.
Applied and Environmental Microbiology
Shiga toxin-producing Escherichia coli (STEC) strains are food-borne pathogens whose ability to produce Shiga toxin (Stx) is due to integration of Stx-encoding lambdoid bacteriophages. These Stx phages are both genetically and morphologically heterogeneous, and here we report the design and validation of a PCR-based multilocus typing scheme. PCR primer sets were designed for database variants of a range of key lambdoid bacteriophage genes and applied to control phages and 70 stx+ phage preparations induced from a collection of STEC isolates. The genetic diversity residing within these populations could be described, and observations were made on the heterogeneity of individual gene targets, including the unexpected predominance of short-tailed phages with a highly conserved tail spike protein gene. Purified Stx phages can be profiled using this scheme, and the lambdoid phage-borne genes in induced STEC preparations can be identified as well as those residing in the noninducible prophage complement. The ultimate goal is to enable robust and realistically applicable epidemiological studies of Stx phages and their traits. The impact of Stx phage on STEC epidemiology is currently unknown.
Related Articles
Identification of genes expressed in cultures of E. coli lysogens carrying the Shiga toxin-encoding prophage Φ24B
Riley, Laura M
Veses-Garcia, Marta
Hillman, Jeffrey D
Handfield, Martin
McCarthy, Alan J
Allison, Heather E
BMC Microbiology
Shigatoxigenic E. coli are a global and emerging health concern. Shiga toxin, Stx, is encoded on the genome of temperate, lambdoid Stx phages. Genes essential for phage maintenance and replication are encoded on approximately 50% of the genome, while most of the remaining genes are of unknown function nor is it known if these annotated hypothetical genes are even expressed. It is hypothesized that many of the latter have been maintained due to positive selection pressure, and that some, expressed in the lysogen host, have a role in pathogenicity. This study used Change Mediated Antigen Technology (CMAT)™ and 2D-PAGE, in combination with RT-qPCR, to identify Stx phage genes that are expressed in E. coli during the lysogenic cycle.
Lysogen cultures propagated for 5-6 hours produced a high cell density with a low proportion of spontaneous prophage induction events. The expression of 26 phage genes was detected in these cultures by differential 2D-PAGE of expressed proteins and CMAT. Detailed analyses of 10 of these genes revealed that three were unequivocally expressed in the lysogen, two expressed from a known lysogenic cycle promoter and one uncoupled from the phage regulatory network.
Propagation of a lysogen culture in which no cells at all are undergoing spontaneous lysis is impossible. To overcome this, RT-qPCR was used to determine gene expression profiles associated with the growth phase of lysogens. This enabled the definitive identification of three lambdoid Stx phage genes that are expressed in the lysogen and seven that are expressed during lysis. Conservation of these genes in this phage genome, and other Stx phages where they have been identified as present, indicates their importance in the phage/lysogen life cycle, with possible implications for the biology and pathogenicity of the bacterial host.
Related Articles
Inhibition of spontaneous induction of lambdoid prophages in Escherichia coli cultures: simple procedures with possible biotechnological applications
Czyz, Agata
Los, Marcin
Wrobel, Borys
Wegrzyn, Grzegorz
BMC Biotechnology
Infections of bacterial cultures by bacteriophages are serious problems in biotechnological laboratories. Apart from such infections, prophage induction in the host cells may also be dangerous. Escherichia coli is a commonly used host in biotechnological production, and many laboratory strains of this bacterium harbour lambdoid prophages. These prophages may be induced under certain conditions leading to phage lytic development. This is fatal for further cultivations as relatively low, though still significant, numbers of phages may be overlooked. Thus, subsequent cultures of non-lysogenic strains may be infected and destroyed by such phage.
Here we report that slow growth of bacteria decreases deleterious effects of spontaneous lambdoid prophage induction. Moreover, replacement of glucose with glycerol in a medium stimulates lysogenic development of the phage after infection of E. coli cells. A plasmid was constructed overexpressing the phage 434 cI gene, coding for the repressor of phage promoters which are necessary for lytic development. Overproduction of the cI repressor abolished spontaneous induction of the λimm434 prophage.
Simple procedures that alleviate problems with spontaneous induction of lambdoid prophage and subsequent infection of E. coli strains by these phages are described. Low bacterial growth rate, replacement of glucose with glycerol in a medium and overproduction of the cI repressor minimise the risk of prophage induction during cultivation of lysogenic bacteria and subsequent infection of other bacterial strains.
Related Articles
Rapid and Accurate Detection of Bacteriophage Activity against Escherichia coli O157:H7 by Propidium Monoazide Real-Time PCR
Liu, Hui
Niu, Yan D.
Li, Jinquan
Stanford, Kim
McAllister, Tim A.
BioMed Research International
Conventional methods to determine the efficacy of bacteriophage (phage) for biocontrol of E. coli require several days, due to the need to culture bacteria. Furthermore, cell surface-attached phage particles may lyse bacterial cells during experiments, leading to an overestimation of phage activity. DNA-based real-time quantitative polymerase chain reaction (qPCR) is a fast, sensitive, and highly specific means of enumerating pathogens. However, qPCR may underestimate phage activity due to its inability to distinguish viable from nonviable cells. In this study, we evaluated the suitability of propidium monoazide (PMA), a microbial membrane-impermeable dye that inhibits amplification of extracellular DNA and DNA within dead or membrane-compromised cells as a means of using qPCR to identify only intact E. coli cells that survive phage exposure. Escherichia coli O157:H7 strain R508N and 4 phages (T5-like, T1-like, T4-like, and O1-like) were studied. Results compared PMA-qPCR and direct plating and confirmed that PMA could successfully inhibit amplification of DNA from compromised/damaged cells E. coli O157:H7. Compared to PMA-qPCR, direct plating overestimated (P < 0.01) phage efficacy as cell surface-attached phage particles lysed E. coli O157:H7 during the plating process. Treatment of samples with PMA in combination with qPCR can therefore be considered beneficial when assessing the efficacy of bacteriophage for biocontrol of E. coli O157:H7.
Related Articles
Insertion of bacteriophage lambda into the deo operon of Escherichia coli K-12 and isolation of plaque-forming lambdadeo+ transducing bacteriophages.
Buxton, R S
Hammer-Jespersen, K
Hansen, T D
Journal of Bacteriology
A procedure has been devised to isolate plaque-forming lambda cI857S7 transducing bacteriophage which carry the internal promoter, P3, of the deo operon of Escherichia coli and the deoB and deoD genes, while lacking the deoP and cytP promoters of the same operon, in order to study, specifically, regulation at the P3 site. This has been accomplished by selecting for the insertion of bacteriophage lambda into the deoA gene in a strain deleted for the normal lambda attachment site (delta att lambda) and isolating from this lysogen lambda spi- and lambda EDTAr phage. Among these, lambda pdeoB+D+ phage were identified by their transducing abilities. From in vivo enzyme induction experiments performed on a delta deo strain lysogenized with such phage, they were shown to carry the P3 promoter while lacking the deoP and cytP promoters. A lambdapdeo B+D+ phage phage was used to lysogenize a deo+ delta att lambda strain, integration of lambda occurring within the region of homology, and, from a heat-induced lysate of this strain, a plaque-forming lambda+ phage carrying the complete deo operon was obtained. Phage lambda was also inserted into the deoB and deoD genes and into the tdk gene. By isolating lambdaspi- and lambdaEDTAr phage from the deo::(lambda) mutants and determining which bacterial genes they carried and whether they retained the int gene of lambda, it was found that lambda had inserted into deoD with the same orientation as lambda inserted into attlambda, whereas lambda inserted into deoA and deoB had the opposite orientation. Deletions extending from the site of lambda insertion into the bacterial chromosome were isolated by selecting for heat-resistant revertants. These confirmed the order of markers to be deo-serB-trpR-thr and also placed a locus, msp, determining sensitivity or resistance of male strains to male-specific phages, between trpR and thr. For some reason unknown, but which may be related to the orientation of the lambda prophages, short deletions rendering the bacterium Ser- Thr+ were of much lower frequency from the deoD::(lambda) lysogen than from the other two lysogens. From an examination of the residual deoD enzyme levels in deoB::(lambda) mutants, it was deduced that there may be two promoter sites within the deoB::(lambda) mutants, it was deduced that there may be two promoter sites within the deoB gene, transcription from one of these being sufficient to account for the noncoordinate nature of the induction of deoB and deoD gene products.
Related Articles
Temperate Phages Acquire DNA from Defective Prophages by Relaxed Homologous Recombination: The Role of Rad52-Like Recombinases
De Paepe, Marianne
Hutinet, Geoffrey
Son, Olivier
Amarir-Bouhram, Jihane
Schbath, Sophie
Petit, Marie-Agnès
PLoS Genetics
Bacteriophages (or phages) dominate the biosphere both numerically and in terms of genetic diversity. In particular, genomic comparisons suggest a remarkable level of horizontal gene transfer among temperate phages, favoring a high evolution rate. Molecular mechanisms of this pervasive mosaicism are mostly unknown. One hypothesis is that phage encoded recombinases are key players in these horizontal transfers, thanks to their high efficiency and low fidelity. Here, we associate two complementary in vivo assays and a bioinformatics analysis to address the role of phage encoded recombinases in genomic mosaicism. The first assay allowed determining the genetic determinants of mosaic formation between lambdoid phages and Escherichia coli prophage remnants. In the second assay, recombination was monitored between sequences on phage λ, and allowed to compare the performance of three different Rad52-like recombinases on the same substrate. We also addressed the importance of homologous recombination in phage evolution by a genomic comparison of 84 E. coli virulent and temperate phages or prophages. We demonstrate that mosaics are mainly generated by homology-driven mechanisms that tolerate high substrate divergence. We show that phage encoded Rad52-like recombinases act independently of RecA, and that they are relatively more efficient when the exchanged fragments are divergent. We also show that accessory phage genes orf and rap contribute to mosaicism. A bioinformatics analysis strengthens our experimental results by showing that homologous recombination left traces in temperate phage genomes at the borders of recently exchanged fragments. We found no evidence of exchanges between virulent and temperate phages of E. coli. Altogether, our results demonstrate that Rad52-like recombinases promote gene shuffling among temperate phages, accelerating their evolution. This mechanism may prove to be more general, as other mobile genetic elements such as ICE encode Rad52-like functions, and play an important role in bacterial evolution itself.
Author Summary
Temperate bacteriophages (or phages) are bacterial viruses that, unlike virulent phages, have the ability to enter a prophage dormant state upon infection, in which they stably replicate with the bacterial genome. A majority of bacterial genomes contain multiple active or defective prophages, and numerous bacterial phenotypes are modified by these prophages, such as increased virulence. These mobile genetic elements are subject to high levels of genetic exchanges, through which new genes are constantly imported into bacterial genomes. Phage-encoded homologous recombination enzymes, or recombinases, are potentially key actors in phage genome shuffling. In this work, we show that gene acquisition in temperate phages is strongly dependent on the presence of sequence homology, but is highly tolerant to divergence. We report that gene exchanges are mainly catalyzed by recombinases found on temperate phages, and show that four different Rad52-like recombinases have a relaxed fidelity in vivo, compared to RecA. This high capacity of exchange speeds up evolution of phages, and indirectly also the evolution of bacteria.
Related Articles
Enumeration of bacteriophage particles
Anderson, Bradley
Rashid, Mohammed H
Carter, Chandi
Pasternack, Gary
Rajanna, Chythanya
Revazishvili, Tamara
Dean, Timothy
Senecal, Andre
Sulakvelidze, Alexander
Bacteriophages are increasingly being utilized and considered for various practical applications, ranging from decontaminating foods and inanimate surfaces to human therapy; therefore, it is important to determine their concentrations quickly and reliably. Traditional plaque assay (PA) is the current "gold standard" for quantitating phage titers. However, it requires at least 18 h before results are obtained, and they may be significantly influenced by various factors. Therefore, two alternative assays based on the quantitative real-time polymerase chain reaction (QPCR) and NanoSight Limited (NS) technologies were recently proposed for enumerating phage particles. The present study compared the three approaches' abilities to quantitate Listeria monocytogenes-, Escherichia coli O157:H7- and Yersinia pestis-specific lytic phages quickly and reproducibly. The average coefficient of variation (CVS) of the PA method including all three phages was 0.15. The reproducibility of the PA method decreased dramatically when multiple investigators performed the assays, and mean differences of as much as 0.33 log were observed. The QPC R method required costly equipment and the synthesis of phage-specific oligonucleotide primers, but it determined phage concentrations faster (within about 4 h) and more precisely than did PA (CVS = 0.13). NS technology required costly equipment, was less precise (CVS = 0.28) than the PA and QPCR methods, and only worked when the phages were suspended in clear medium. However, it provided results within 5 min. After the overall correlation is established with the PA method, either of the two assays may be useful for quickly and reproducibly determining phage concentrations.
bacteriophage; phage; plaque assays; phage titer
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Manipulating or Superseding Host Recombination Functions: A Dilemma That Shapes Phage Evolvability
Bobay, Louis-Marie
Touchon, Marie
Rocha, Eduardo P. C.
PLoS Genetics
Phages, like many parasites, tend to have small genomes and may encode autonomous functions or manipulate those of their hosts'. Recombination functions are essential for phage replication and diversification. They are also nearly ubiquitous in bacteria. The E. coli genome encodes many copies of an octamer (Chi) motif that upon recognition by RecBCD favors repair of double strand breaks by homologous recombination. This might allow self from non-self discrimination because RecBCD degrades DNA lacking Chi. Bacteriophage Lambda, an E. coli parasite, lacks Chi motifs, but escapes degradation by inhibiting RecBCD and encoding its own autonomous recombination machinery. We found that only half of 275 lambdoid genomes encode recombinases, the remaining relying on the host's machinery. Unexpectedly, we found that some lambdoid phages contain extremely high numbers of Chi motifs concentrated between the phage origin of replication and the packaging site. This suggests a tight association between replication, packaging and RecBCD-mediated recombination in these phages. Indeed, phages lacking recombinases strongly over-represent Chi motifs. Conversely, phages encoding recombinases and inhibiting host recombination machinery select for the absence of Chi motifs. Host and phage recombinases use different mechanisms and the latter are more tolerant to sequence divergence. Accordingly, we show that phages encoding their own recombination machinery have more mosaic genomes resulting from recent recombination events and have more diverse gene repertoires, i.e. larger pan genomes. We discuss the costs and benefits of superseding or manipulating host recombination functions and how this decision shapes phage genome structure and evolvability.
Author Summary
Bacterial viruses, called bacteriophages, are extremely abundant in the biosphere. They have key roles in the regulation of bacterial populations and in the diversification of bacterial genomes. Among these viruses, lambdoid phages are very abundant in enterobacteria and exchange genetic material very frequently. This latter process is thought to increase phage diversity and therefore facilitate adaptation to hosts. Recombination is also essential for the replication of many lambdoid phages. Lambdoids have been described to encode their own recombination genes and inhibit their hosts'. In this study, we show that lambdoids are split regarding their capacity to encode autonomous recombination functions and that this affects the abundance of recombination-related sequence motifs. Half of the phages encode an autonomous system and inhibit their hosts'. The trade-off between superseding and manipulating the hosts' recombination functions has important consequences. The phages encoding autonomous recombination functions have more diverse gene repertoires and recombine more frequently. Viruses, as many other parasites, have small genomes and depend on their hosts for several housekeeping functions. Hence, they often face trade-offs between supersession and manipulation of molecular machineries. Our results suggest these trade-offs may shape viral gene repertoires, their sequence composition and even influence their evolvability.
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The CI Repressors of Shiga Toxin-Converting Prophages Are Involved in Coinfection of Escherichia coli Strains, Which Causes a Down Regulation in the Production of Shiga Toxin 2▿
Serra-Moreno, R.
Jofre, J.
Muniesa, M.
Journal of Bacteriology
Shiga toxins (Stx) are the main virulence factors associated with a form of Escherichia coli known as Shiga toxin-producing E. coli (STEC). They are encoded in temperate lambdoid phages located on the chromosome of STEC. STEC strains can carry more than one prophage. Consequently, toxin and phage production might be influenced by the presence of more than one Stx prophage on the bacterial chromosome. To examine the effect of the number of prophages on Stx production, we produced E. coli K-12 strains carrying either one Stx2 prophage or two different Stx2 prophages. We used recombinant phages in which an antibiotic resistance gene (aph, cat, or tet) was incorporated in the middle of the Shiga toxin operon. Shiga toxin was quantified by immunoassay and by cytotoxicity assay on Vero cells (50% cytotoxic dose). When two prophages were inserted in the host chromosome, Shiga toxin production and the rate of lytic cycle activation fell. The cI repressor seems to be involved in incorporation of the second prophage. Incorporation and establishment of the lysogenic state of the two prophages, which lowers toxin production, could be regulated by the CI repressors of both prophages operating in trans. Although the sequences of the cI genes of the phages studied differed, the CI protein conformation was conserved. Results indicate that the presence of more than one prophage in the host chromosome could be regarded as a mechanism to allow genetic retention in the cell, by reducing the activation of lytic cycle and hence the pathogenicity of the strains.
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ppGpp-Dependent Negative Control of DNA Replication of Shiga Toxin-Converting Bacteriophages in Escherichia coli
Nowicki, Dariusz
Kobiela, Wioletta
Węgrzyn, Alicja
Węgrzyn, Grzegorz
Szalewska-Pałasz, Agnieszka
Journal of Bacteriology
The pathogenicity of enterohemorrhagic Escherichia coli (EHEC) strains depends on the production of Shiga toxins that are encoded on lambdoid prophages. Effective production of these toxins requires prophage induction and subsequent phage replication. Previous reports indicated that lytic development of Shiga toxin-converting bacteriophages is inhibited in amino acid-starved bacteria. However, those studies demonstrated that inhibition of both phage-derived plasmid replication and production of progeny virions occurred during the stringent as well as the relaxed response to amino acid starvation, i.e., in the presence as well as the absence of high levels of ppGpp, an alarmone of the stringent response. Therefore, we asked whether ppGpp influences DNA replication and lytic development of Shiga toxin-converting bacteriophages. Lytic development of 5 such bacteriophages was tested in an E. coli wild-type strain and an isogenic mutant that does not produce ppGpp (ppGpp0). In the absence of ppGpp, production of progeny phages was significantly (in the range of an order of magnitude) more efficient than in wild-type cells. Such effects were observed in infected bacteria as well as after prophage induction. All tested bacteriophages formed considerably larger plaques on lawns formed by ppGpp0 bacteria than on those formed by wild-type E. coli. The efficiency of synthesis of phage DNA and relative amount of lambdoid plasmid DNA were increased in cells devoid of ppGpp relative to bacteria containing a basal level of this nucleotide. We conclude that ppGpp negatively influences the lytic development of Shiga toxin-converting bacteriophages and that phage DNA replication efficiency is limited by the stringent control alarmone.
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Bacteriophage Crosstalk: Coordination of Prophage Induction by Trans-Acting Antirepressors
Lemire, Sébastien
Figueroa-Bossi, Nara
Bossi, Lionello
PLoS Genetics
Many species of bacteria harbor multiple prophages in their genomes. Prophages often carry genes that confer a selective advantage to the bacterium, typically during host colonization. Prophages can convert to infectious viruses through a process known as induction, which is relevant to the spread of bacterial virulence genes. The paradigm of prophage induction, as set by the phage Lambda model, sees the process initiated by the RecA-stimulated self-proteolysis of the phage repressor. Here we show that a large family of lambdoid prophages found in Salmonella genomes employs an alternative induction strategy. The repressors of these phages are not cleaved upon induction; rather, they are inactivated by the binding of small antirepressor proteins. Formation of the complex causes the repressor to dissociate from DNA. The antirepressor genes lie outside the immunity region and are under direct control of the LexA repressor, thus plugging prophage induction directly into the SOS response. GfoA and GfhA, the antirepressors of Salmonella prophages Gifsy-1 and Gifsy-3, each target both of these phages' repressors, GfoR and GfhR, even though the latter proteins recognize different operator sites and the two phages are heteroimmune. In contrast, the Gifsy-2 phage repressor, GtgR, is insensitive to GfoA and GfhA, but is inactivated by an antirepressor from the unrelated Fels-1 prophage (FsoA). This response is all the more surprising as FsoA is under the control of the Fels-1 repressor, not LexA, and plays no apparent role in Fels-1 induction, which occurs via a Lambda CI-like repressor cleavage mechanism. The ability of antirepressors to recognize non-cognate repressors allows coordination of induction of multiple prophages in polylysogenic strains. Identification of non-cleavable gfoR/gtgR homologues in a large variety of bacterial genomes (including most Escherichia coli genomes in the DNA database) suggests that antirepression-mediated induction is far more common than previously recognized.
Author Summary
Many viruses that infect bacteria (bacteriophages) can direct the integration of their DNA into the bacterial chromosome. This condition, known as lysogeny, is relevant to bacterial evolution, as it is one of the main pathways leading to the incorporation of foreign DNA in nature. Indeed, bacteriophages often carry genes that escape lysogenic repression and benefit the bacterium. This symbiotic association can come to an end if bacteria suffer DNA damage. A mechanism mediated by the host's RecA protein causes the relief of repression, viral DNA excision, and replication. This process, known as prophage induction, kills the host and results in the release of viral particles. In this work, we have analyzed the mechanism responsible for induction in a large family of prophages naturally present in the genomes of Salmonella bacteria. We found that, unlike in best-studied model phages, the repressors of these Salmonella phages do not undergo RecA-mediated proteolysis; rather, they are inactivated by the binding of small antirepressor proteins. We show that some antirepressors can act on both cognate and non-cognate repressors, allowing separate prophages within a given strain to be induced simultaneously. We discuss evidence suggesting that antirepressor-mediated prophage induction is quite common in the bacterial world.
Related Articles
Comparative genomics of Shiga toxin encoding bacteriophages
Smith, Darren L
Rooks, David J
Fogg, Paul CM
Darby, Alistair C
Thomson, Nick R
McCarthy, Alan J
Allison, Heather E
BMC Genomics
Stx bacteriophages are responsible for driving the dissemination of Stx toxin genes (stx) across their bacterial host range. Lysogens carrying Stx phages can cause severe, life-threatening disease and Stx toxin is an integral virulence factor. The Stx-bacteriophage vB_EcoP-24B, commonly referred to as Ф24B, is capable of multiply infecting a single bacterial host cell at a high frequency, with secondary infection increasing the rate at which subsequent bacteriophage infections can occur. This is biologically unusual, therefore determining the genomic content and context of Ф24B compared to other lambdoid Stx phages is important to understanding the factors controlling this phenomenon and determining whether they occur in other Stx phages.
The genome of the Stx2 encoding phage, Ф24B was sequenced and annotated. The genomic organisation and general features are similar to other sequenced Stx bacteriophages induced from Enterohaemorrhagic Escherichia coli (EHEC), however Ф24B possesses significant regions of heterogeneity, with implications for phage biology and behaviour. The Ф24B genome was compared to other sequenced Stx phages and the archetypal lambdoid phage, lambda, using the Circos genome comparison tool and a PCR-based multi-loci comparison system.
The data support the hypothesis that Stx phages are mosaic, and recombination events between the host, phages and their remnants within the same infected bacterial cell will continue to drive the evolution of Stx phage variants and the subsequent dissemination of shigatoxigenic potential.
Related Articles
Type III Restriction Is Alleviated by Bacteriophage (RecE) Homologous Recombination Function but Enhanced by Bacterial (RecBCD) Function
Handa, Naofumi
Kobayashi, Ichizo
Journal of Bacteriology
Previous works have demonstrated that DNA breaks generated by restriction enzymes stimulate, and are repaired by, homologous recombination with an intact, homologous DNA region through the function of lambdoid bacteriophages lambda and Rac. In the present work, we examined the effect of bacteriophage functions, expressed in bacterial cells, on restriction of an infecting tester phage in a simple plaque formation assay. The efficiency of plaque formation on an Escherichia coli host carrying EcoRI, a type II restriction system, is not increased by the presence of Rac prophage—presumably because, under the single-infection conditions of the plaque assay, a broken phage DNA cannot find a homologue with which to recombine. To our surprise, however, we found that the efficiency of plaque formation in the presence of a type III restriction system, EcoP1 or EcoP15, is increased by the bacteriophage-mediated homologous recombination functions recE and recT of Rac prophage. This type III restriction alleviation does not depend on lar on Rac, unlike type I restriction alleviation. On the other hand, bacterial RecBCD-homologous recombination function enhances type III restriction. These results led us to hypothesize that the action of type III restriction enzymes takes place on replicated or replicating DNA in vivo and leaves daughter DNAs with breaks at nonallelic sites, that bacteriophage-mediated homologous recombination reconstitutes an intact DNA from them, and that RecBCD exonuclease blocks this repair by degradation from the restriction breaks.
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The Generalized Transducing Salmonella Bacteriophage ES18: Complete Genome Sequence and DNA Packaging Strategy
Casjens, Sherwood R.
Gilcrease, Eddie B.
Winn-Stapley, Danella A.
Schicklmaier, Petra
Schmieger, Horst
Pedulla, Marisa L.
Ford, Michael E.
Houtz, Jennifer M.
Hatfull, Graham F.
Hendrix, Roger W.
Journal of Bacteriology
The generalized transducing double-stranded DNA bacteriophage ES18 has an icosahedral head and a long noncontractile tail, and it infects both rough and smooth Salmonella enterica strains. We report here the complete 46,900-bp genome nucleotide sequence and provide an analysis of the sequence. Its 79 genes and their organization clearly show that ES18 is a member of the lambda-like (lambdoid) phage group; however, it contains a novel set of genes that program assembly of the virion head. Most of its integration-excision, immunity, Nin region, and lysis genes are nearly identical to those of the short-tailed Salmonella phage P22, while other early genes are nearly identical to Escherichia coli phages λ and HK97, S. enterica phage ST64T, or a Shigella flexneri prophage. Some of the ES18 late genes are novel, while others are most closely related to phages HK97, lambda, or N15. Thus, the ES18 genome is mosaically related to other lambdoid phages, as is typical for all group members. Analysis of virion DNA showed that it is circularly permuted and about 10% terminally redundant and that initiation of DNA packaging series occurs across an approximately 1-kbp region rather than at a precise location on the genome. This supports a model in which ES18 terminase can move substantial distances along the DNA between recognition and cleavage of DNA destined to be packaged. Bioinformatic analysis of large terminase subunits shows that the different functional classes of phage-encoded terminases can usually be predicted from their amino acid sequence.
Related Articles
Single-cell and population level viral infection dynamics revealed by phageFISH, a method to visualize intracellular and free viruses
Allers, Elke
Moraru, Cristina
Duhaime, Melissa B
Beneze, Erica
Solonenko, Natalie
Barrero-Canosa, Jimena
Amann, Rudolf
Sullivan, Matthew B
Environmental Microbiology
Microbes drive the biogeochemical cycles that fuel planet Earth, and their viruses (phages) alter microbial population structure, genome repertoire, and metabolic capacity. However, our ability to understand and quantify phage–host interactions is technique-limited. Here, we introduce phageFISH – a markedly improved geneFISH protocol that increases gene detection efficiency from 40% to > 92% and is optimized for detection and visualization of intra- and extracellular phage DNA. The application of phageFISH to characterize infection dynamics in a marine podovirus–gammaproteobacterial host model system corroborated classical metrics (qPCR, plaque assay, FVIC, DAPI) and outperformed most of them to reveal new biology. PhageFISH detected both replicating and encapsidated (intracellular and extracellular) phage DNA, while simultaneously identifying and quantifying host cells during all stages of infection. Additionally, phageFISH allowed per-cell relative measurements of phage DNA, enabling single-cell documentation of infection status (e.g. early vs late stage infections). Further, it discriminated between two waves of infection, which no other measurement could due to population-averaged signals. Together, these findings richly characterize the infection dynamics of a novel model phage–host system, and debut phageFISH as a much-needed tool for studying phage–host interactions in the laboratory, with great promise for environmental surveys and lineage-specific population ecology of free phages.
Related Articles
The Lysis-Lysogeny Decision of Bacteriophage 933W: a 933W Repressor-Mediated Long-Distance Loop Has No Role in Regulating 933W PRM Activity▿
Bullwinkle, Tammy J.
Koudelka, Gerald B.
Journal of Bacteriology
Our data show that unlike bacteriophage λ, repressor bound at OL of bacteriophage 933W has no role in regulation of 933W repressor occupancy of 933W OR3 or the transcriptional activity of 933W PRM. This finding suggests that a cooperative long-range loop between repressor tetramers bound at OR and OL does not form in bacteriophage 933W. Nonetheless, 933W forms lysogens, and 933W prophage display a threshold response to UV induction similar to related lambdoid phages. Hence, the long-range loop thought to be important for constructing a threshold response in lambdoid bacteriophages is dispensable. The lack of a loop requires bacteriophage 933W to use a novel strategy in regulating its lysis-lysogeny decisions. As part of this strategy, the difference between the repressor concentrations needed to bind OR2 and activate 933W PRM transcription or bind OR3 and repress transcription from PRM is <2-fold. Consequently, PRM is never fully activated, reaching only ∼25% of the maximum possible level of repressor-dependent activation before repressor-mediated repression occurs. The 933W repressor also apparently does not bind cooperatively to the individual sites in OR and OL. This scenario explains how, in the absence of DNA looping, bacteriophage 933W displays a threshold effect in response to DNA damage and suggests how 933W lysogens behave as "hair triggers" with spontaneous induction occurring to a greater extent in this phage than in other lambdoid phages.
Related Articles
Immunity Profiles of Wild-Type and Recombinant Shiga-Like Toxin-Encoding Bacteriophages and Characterization of Novel Double Lysogens
Allison, Heather E.
Sergeant, Martin J.
James, Chloë E.
Saunders, Jon R.
Smith, Darren L.
Sharp, Richard J.
Marks, Trevor S.
McCarthy, Alan J.
Infection and Immunity
The pathogenicity of Shiga-like toxin (stx)-producing Escherichia coli (STEC), notably serotype O157, the causative agent of hemorrhagic colitis, hemolytic-uremic syndrome, and thrombotic thrombocytopenic purpura, is based partly on the presence of genes (stx1 and/or stx2) that are known to be carried on temperate lambdoid bacteriophages. Stx phages were isolated from different STEC strains and found to have genome sizes in the range of 48 to 62 kb and to carry either stx1 or stx2 genes. Restriction fragment length polymorphism patterns and sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein profiles were relatively uninformative, but the phages could be differentiated according to their immunity profiles. Furthermore, these were sufficiently sensitive to enable the identification and differentiation of two different phages, both carrying the genes for Stx2 and originating from the same STEC host strain. The immunity profiles of the different Stx phages did not conform to the model established for bacteriophage lambda, in that the pattern of individual Stx phage infection of various lysogens was neither expected nor predicted. Unexpected differences were also observed among Stx phages in their relative lytic productivity within a single host. Two antibiotic resistance markers were used to tag a recombinant phage in which the stx genes were inactivated, enabling the first reported observation of the simultaneous infection of a single host with two genetically identical Stx phages. The data demonstrate that, although Stx phages are members of the lambdoid family, their replication and infection control strategies are not necessarily identical to the archetypical bacteriophage λ, and this could be responsible for the widespread occurrence of stx genes across a diverse range of E. coli serotypes.
Related Articles
Quantification and Evaluation of Infectivity of Shiga Toxin-Encoding Bacteriophages in Beef and Salad ▿
Imamovic, Lejla
Muniesa, Maite
Applied and Environmental Microbiology
Stx bacteriophages in 68 samples of beef and salad were quantified by real-time quantitative PCR (qPCR). Stx phages from the samples were propagated in Escherichia coli C600, E. coli O157:H7, and Shigella strains and further quantified. Fifty percent of the samples carried infectious Stx phages that were isolated from plaques generated by lysis.
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Team aims to develop nuclear detector using noble gases
Plans for a new radiation detector to help stop the illegal supply of nuclear materials could address the growing costs of current technology.
Researchers at Liverpool University are developing a device that relies on xenon and other noble gases instead of an increasingly rare type of helium used in some existing equipment to detect neutron radiation, as part of a €3.3m (£2.7m) EU-funded project.
The academics, working with other universities and commercial partners across Europe, plan to produce a prototype device to help catch smugglers and terrorists handling nuclear materials that could be used to make weapons.
The price of the helium-3 used in current detectors has gone up by a factor of 10 in the last few years, said Liverpool's research leader Prof Christos Touramanis.
'Helium-3 is a by-product of the nuclear weapons industry and, as people are not making more plutonium because there's no need for it, the stockpiles are running out,' he told The Engineer.
In existing detectors, neutrons collide with the nuclei of helium-3 atoms to produce light at a specific wavelength, which can then be used to identify the source of the neutrons via computer algorithms that analyse the light spectrum.
Touramanis said there were two novel requirements of the research: adapting the technology to work with other noble gases and turning it into a simple, rugged device for use in the field.
'If you take a detector and just put some noble gas in you will not get out what you want. You need to play with the parameters of the operation [such as] the different wavelengths and amounts of light.
'The second difference is that most radiation detectors, including those with helium-3, were made for laboratory use and so are not rugged enough for putting in the back of a van.
'Also, between the detector itself and the computers you need to read it, you end up with something big and heavy that requires expert operators.'
The researchers want the final device, which will be integrated and assembled in Liverpool towards the end of the three-and-a-half year project, to comprise a modular system of small elements, including a simple traffic light-style indicator of whether nuclear materials are present.
Oxford University and Imperial College London are undertaking a similar project that is using the isotope lithium-6 in place of helium-3.
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The Miami Hurricanes have been the unexpected talk of the upcoming NFL Draft; a balance of both some positive and negative news.
The good; a handful of quality Canes look to dominate this year's selection process and the next crop of kids to join the NFL U ranks are here.
The not-so-good? The fact that Al Golden and his staff ended the season…
Former Miami Hurricanes cornerback Antonio Crawford is reportedly transferring to West Virginia and it conjured up the philosophical thought experiment regarding a tree falling in the forest and whether it makes a sound if no one is around to hear it.
If a former Canes athlete who smack-talked the program decides to transfer after an average-at-best run during in his two years…
The Miami Hurricanes woke from a slumber and gave the Florida State Seminoles the type of Sunday beating that fans live for in this rivalry; a convincing, 10-0 shellacking that reminded everyone just what this team can do when it's firing on all cylinders.
Unfortunately, Miami's familiar alter ego surfaced on Saturday—a team hungover and beaten down from Friday night's heartbreaking…
Below is a guest piece from columnist Scott Salomon, who attended Friday's night's unveiling of the statue, honoring the late, great, legendary Ron Fraser, en route to Miami's extra-innings loss to rival Florida State. Look for more work here at allCanesBlog.com from Scott in the future.
CORAL GABLES — More than 25 years ago Liz Fraser-Kraut was a valued employee…
It didn't really have all the fanfare and hype of National Signing Day and watching a 5-star recruit pull a U-emblazoned hat out of a bag when choosing Miami, but in all reality it could prove to be the Canes' biggest boom-or-bust "recruit" in years.
Harvard health dean Julio Frenk was named the sixth president in University of Miami history days back…
Listen LIVE every Thursday from
7-9pm ET at allCanes.com or stream live through the allCanes iPhone app allCanes Radio will again be broadcasting live from Titanic Restaurant & Brewery, next door to allCanes.
Miss a show? Check out the
program archive online."
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Saturday: 10am - 6pm
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The Big E GaffneyRead MoreThe Big E ? Entertainment Outlet combines the joys of a movie theater, bowling alley, indoor playground, and full arcade into one location. The full movie theater shows the latest releases on eight screens. The bowling alley hosts both league and public play, as well as dedicating every Friday and Saturday night to proper cosmic bowling events where they turn down the lights, turn up the music, and chuck bowling balls at the stars to try to knock them from the sky. The FunE Business Playland and Arcade occupies the rest of the complex, combining an indoor playground with an arcade. Here, the playground features slides, climbing equipment, and a Ball-O-Sity Blast Center where kids can shoot foam balls from little cannons, running and playing in an environment designed specifically with safety in mind. They can also take the bumper cars for a spin. The arcade, on the other hand, challenges visitors with tandem vehicle racing, Guitar Hero, and other modern favorites, as well as classic installations such as ski ball. Like at any good arcade, the games dispense tickets based on performance, and electronic points can be exchanged for prizes including MP3 players, All-Terrain Vehicles, or an equal number of tickets in a different color.1100 Factory Shops BoulevardGaffney, SC US
Regal Manor TwinRead MoreWith an arsenal of informative magazines, elegant photographs, and illuminating documentaries, National Geographic has inspired planetary responsibility and natural wonderment for more than 120 years. Their latest filmed adventure, The Last Lions, ushers viewers into the wetlands of Botswana's Okavango Delta, where a lioness named Ma di Tau and her cubs fight for their survival. From fleeing raging fires and cub-killing rival prides to wading through crocodile-infested rivers and the supermarket at rush hour, this family suffers perils that leave audiences touched and awestruck. Crafted by award-winning filmmakers, Dereck and Beverly Joubert, and narrated by Jeremy Irons, The Last Lions aims to raise awareness of dwindling big-cat populations while sharing a compelling story of hope. The film is rated PG for depictions of the food-chain cycle without the accompaniment of an Elton John song.607 Providence RdCharlotte, NC US
Ayrsley Grand Cinema 14Read MoreThe Ayrsley Grand Theater is a local landmark in Charlotte, North Carolina. They've been voted the best theater in the state multiple times, the most recent being in 2012. They feature comfortable, breathable seating that's maintained to an exceptional level of cleanliness. They also have 3D projectors and glasses for 3D feature films. Unlike other movie theaters that are simply too nippy, the Ayrsley is kept at just the right temperature. Arguably, the best part of seeing a movie at the Ayrsley is their $5 matinees, which are accommodatingly timed before 4PM on any day of the week. There are plenty of fine dining options surrounding the Ayrsley, making it the perfect place for a date.9110 Kings Parade BlvdCharlotte, NC US
CinebarreRead MoreSpice up your week with a trip to Cinebarre in Charlotte, and enjoy grub at the top of its game. Tots and tykes will be right at home at Cinebarre with its kid-approved food and ambience. If dinner conversation isn't your thing, Cinebarre's got you covered with live tunes from a DJ or musical group. DJ fans will appreciate Cinebarre's frequent live mixes. Patrons have the pleasure of listening to live music while they dine. Interested in eating out over the weekend? Keep in mind that the restaurant gets swamped on Fridays and Saturdays, and service may take longer than expected. Easily accessible parking options are located near this dining establishment.8008 Providence RoadCharlotte, NC US
Regal Park Terrace Stadium 6Read MoreFor the movies you can't see anywhere else, you have to visit Regal Park Terrace Stadium 6. The theater hosts independent movies in a clean and friendly setting. Enjoy a movie in a theater offering traditional concessions, six movie screens, and comfortable seats. At Regal Park Terrace Stadium 6, you can watch blockbuster favorites and brand-new indie releases for less than what you'd pay at other theaters. You'll also find something a little different, like healthy snacks, and a welcoming, professional staff. When your movie selection (and experience) deserves a change, the Regal Park Terrace Stadium 6 has just what you need.4289 Park RdCharlotte, NC US


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We came here thinking it would be an interesting tasty last dinner in Morocco before we flew back the next day. The restaurant was empty, except for the owner who filled the restaurant with cigarette smoke whilst standing at the bar doing nothing. We ordered a couple of burgers. Which were so close to being good but yet so far. They were raw! and the chips covered in masses of salt. We sent them back and received the very same chips and bun with the same burger which had just been put back on the grill. A very poor effort considering the restaurant was empty! We were offered no discount on bill despite complaints. A poor experience all round. Much better places to go in this area of Marrakech.
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News from the Central region
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Margaret Dougher Lewandowski, former resident of Pittston, passed away Sunday, Oct. 7, 2012 at Meadows Nursing Home, Dallas.
Born Feb. 15, 1920 in Duryea, she was the daughter of the late John Langhorne and Elizabeth Langhorne Carlson. Margaret attended the Duryea school system and graduated from the Hoyt School of Practical Nursing and the Robert Packer School of Surgical Technology in Sayre.
She was employed by Taylor Hospital, Taylor, as a practical nurse and operating room technician.
Margaret volunteered for the retired senior volunteer project. She worked blood drives and feed a friend, taught arthritis self-help courses at the Kirby Center and helped seniors with their taxes. She helped start the Avoca Library, volunteered at Nesbitt Memorial Hospital, Salvation Army and the American Red Cross. She was a member and past president of the Avoca Women's Club. She was a proud member of the Polka Jets and Dance Troup which entertained at senior centers.
She was also a member of the
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Ladies Auxilary, Avoca.
Surviving are a daughter, Peggy Dougher Rave and husband, Robert, Dallas; three grandchildren, John Metz and wife, Roberta, Atlanta, Georgia; Jeffrey Metz and wife, Susan, Dallas; and Maureen Gallagher, Dallas; three great-grandchildren, Kaitlyn, Ashley and Jeffrey Metz; numerous nieces and nephews; and a brother, Oscar Langhorne, Georgia.
She was preceded in death by her husband, Frank Lewandowski; three brothers, Rudolph, John, and Thomas; and a sister, Emma.
She was also preceded in death by infant son, Joseph.
The funeral will be at 9 a.m. Thursday from the Paul F. Leonard Funeral Home, 575 N. Main St., Pittston, with a Mass of Christian Burial at 9:30 a.m. in Our Lady of the Eucharist Parish, Pittston. Interment will be in St. Mary, Help of Christians Cemetery, Pittston. Friends may call from 5 to 8 p.m. Wednesday.
The family would like to give special thanks to the staff at the Meadows Nursing Home for their kindness, care, and love they provided for "Gram."
In lieu of flowers donations may be sent to the American Red Cross, 256 N. Sherman St., Wilkes-Barre, PA 18702.
Published in Citizens' Voice on Oct. 8, 2012
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Versatile Fox Red Lab in Wisconsin - Labrador Retriever Hunting Dog Pictures
Breed: Labrador Retriever
From: Aaron Palbrach
Website: http://www.iowabirddogs.com
Posted: Jan 14, 2013
This is Kale, a 1 1/2 year old Fox Red labrador retriever out of Bear Creek Kennels in Marengo, Iowa.
She has been a tremendous dog thus far, hunting upland and waterfowl and even trained for shed hunting here in Wisconsin. She is a fantastic dog.
She has completed her AKC Junior Title this past year and we look forward to further competitions and hunting in the future.


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Yoga for Depression by Cormac Lennon Yoga Psychology Yoga Psychology is preventative-curative-promotive. Yoga Psychology is the bridge between the disciplines of Yoga and Psychology. Yoga Psychology is a branch of Science that aims to provide amicable adjustment and promotion of the self by the study and application of Yogic Methods and Models, principles and techniques. It's a Science because its methods are scientific and can be verified. It aims for amicable adjustment - every day we face situations that call for adjustments within ourselves. If we cannot adjust to the given situations, this leads to imbalance. Promotion of self - this involves our quality of life - Yoga Psychology takes this level of quality to a higher level - we learn how to lessen the negative aspects and increase the positive aspects of our personality. A comparison of both negative and positive aspects determines the quality of our lives. Yoga is both a Basic Science, which provides the theories and methods of attaining a balanced personality, and it is an Applied Science. This is the actual application of the methods provided. It aims to re establish balance; it provides tools in problem management. Application of Yogic principles According to Yoga, a person is healthy if he/she has: The energy to cope with daily tasks. A quiet and peaceful mind. A sense of oneness. This can be attained through control of mental modifications. The mental modifications which disturb the personality have their roots in "Avidja" (Ignorance) a false image of ourselves. This is the root of all our sorrows. Through Yoga we can develop: Positive attitude: feelings and emotions. Improve mental potential: intelligence, creativity and problem solving. Balance with the physical system. Combating social problems: from the individual to society. When Yogic principles are applied, it brings an end to conflict. Promotion of self: leads us towards enlightenment. Page 1 of 3 Yoga for Depression by Cormac Lennon The Methods The first, principal method is INTROSPECTION - LOOKING WITHIN, Asana, (Positive), Pranayma (Breathing Techniques) are all sub divisions Of INTROSPECTION. While performing either the person is required to look within and observe the effects. Within these, there are 3 essential aspects. Visualization - non involvement of the senses Awareness - from physical to mental (this leads to a separation from the physical and mental bodies). Becoming a Witness - a direct result of the two previous. Yoga is a useful practice that can bring us back to a balanced state, to a state of being where we can cope with daily living, where we are given tools that will enable us to deal with problems such as depression. Almost everybody will or has experienced depression at some stage of their life. It's not being able to overcome this depression that leads to deeper depression, and once you are caught in a state of deep clinical depression, it may seem difficult or even impossible to get out. We can only begin on the path of Yoga from where we are now, and if that is in a depressed state, then so be it. Our weaknesses are truly our strengths. Yoga has been misunderstood here in the West. It has nothing to do with religion or culture. It is a Science, and it is applicable to all regardless of age, race or creed. It is the "art of living." It is also more than just posture and breathing exercises, it is away of being, it gives us discipline in our lives. If a river has no banks, its water will be dissipated on both sides, and it will never reach its destination of the sea. So, too, it is with ourselves. Yoga can be the banks of our lives, guiding us to reach our own destination, whatever that may be. The Yogic Model for living is D.A.I.L.Y. This stands for: Diet Attitude Life-style Yoga Practice This Yogic Model is applicable to all states of being. So, from this Model, the first aspect to look at is: DIET: We are what we eat literally; the body takes what it needs from our food, expels what it doesn't need and turns the rest back into itself. The body is constantly renewing itself through this means, Page 2 of 3 Yoga for Depression by Cormac Lennon so if we have a bad diet, it will manifest itself in the body. This, in turn, will affect our mental attitude and indeed, affects the whole person. ATTITUDE: How we think and feel about ourselves. Just by changing our attitudes, we can change our lives. Nothing is achieved is life without concentration and a proper attitude. If we feel that we can overcome our problems, then that attitude, if maintained, will lead us to achieving our goals. This aspect of the Yogic Model cannot be underestimated. LIFE STYLE Look around you, look at your environment your, house/flat, the people you associate with. These are all reflections on you and your lifestyle. What you do and who you do it with says a lot about who we are and what style of life we have. If you are not feeling your best and feel negative and cynical towards people around you and nothing seems right then yogic practices may give you the initial boost to carry you forward. Basically there are two kinds of mental disorders: anxiety and depressive disorders. Both need separate practices. The Central Nervous System is divided into 2 Sympathetic and the Parasympathetic. In anxiety disorders, the Sympathetic System is dominant the body is in almost constant stress. The Yogic approach to this disorder is calming and cooling practices and parasympathetic arousal. In Depressive Disorder, sympathetic arousal is applied and dynamic practices. Both these approaches lead to balance and greater harmony. Practices for depression and anxiety include dynamic postures, backbends, standing and twisting; breathing techniques including abdominal breathing. Asana or posture: These have a direct effect on the Endocrine System and the Central Nervous System, balancing and toning both This in turn has an effect on the emotions. It leads to mental and emotional equanimity, practiced with awareness it relieves stress and worry and it also leads to freedom of the personality. Asana relaxes the body, making it light and supple. We're left feeling calm and confident, and we become masters of our own bodies. Flexibility of body leads to a flexible mind, rigidity of body is a rigid mind. Prasnayama (Breathing Techniques) "Life is the period between 1 breath and the next. He who breathes correctly acquires control of the whole being. " Breath is the key link between body and mind. It is intimately connected with every cell in our body. To establish regular rhythmic patterns breaks down negative patterns. It increases absorption of Prana (Life Force), boosting the Immune System and overall well being. It influences the Nervous System in a positive way. Page 3 of 3
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The food was excellent with a wonderful selection. The price on weekday dinner is $15.99 (September 2013) which is an excellent value! The service was great and I would definitely eat here again. They close from 3pm-4pm daily.
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Updated and enhanced with the latest Balanced Scorecard topics, this influential book empowers your organization to turn strategy into performance at every organizational level and translate your intangible resources, such as innovation, customer relationships, and intellectual capital, into real value. The Second Edition of Balanced Scorecard Step-by-Step includes updated case studies as well as new and expanded coverage on:
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Adquirí este libro y no puede parar de leerlo hasta que lo terminé. Simple, bien escrito, práctico. Read morePublished on Aug. 21 2003
If you are interested in the experience of companies that implemented the BSC, read the Kaplan and Norton books. Read morePublished on Aug. 7 2003 by MAURICIO AGUIAR
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Pro Activ 3-Step System
3402 reviews
34% would repurchase
Package Quality: 3.2
Price: $$$
Package Quality: 3.2
Price: $$$
on 7/30/2008 10:06:00 AM
Age: 19-24
Skin: Combination, Fair-Medium, Not Sure
Hair: Brunette, Wavy, Coarse
Eyes: Green
I used this for about a year and it worked great for me! But after a year, my skin started retreating back to its acne-prone combination skin. I stopped using this and switched to Philosophy's Acne Control Kit from Sephora and have been using that since and it works well for me!
on 7/29/2008 11:19:00 PM
Age: 25-29
Skin: Oily, Fair-Medium, Not Sure
Hair: Blond, Other, Other
Eyes: Green
I really wanted to like this product after my friends who used to have acne, after raving about Pro Activ, now have clear, beautiful skin. While I don't have that bad of acne, I still get pimples every so often, though they are easy to hide and go away quickly. I guess I wanted to try something which was supposed to be a "miracle worker" to make my pimples gone for good. So I ordered it, and was only left with worse skin, irritation, burning, stinging, itching, drying, etc. It actually made me break out more. I went to the dermatologist right away and she said she has seen it work for some people, but not all. She put me on an rx (Benzaclin, which I LOVE!) but it still took a few weeks to get my skin back to how it was. When I called to stop Pro Activ from sending more of the product, the woman started questioning me about why I wanted to stop using it. She said to give it at least 2 months (even though my skin was red and raw), however when I refused and told her I just wanted to return it, she obliged. Since then, I have still heard success stories from Pro Activ from my roommate, and other people. I guess it works great for some people, and not at all for others (like me!)
on 7/29/2008 8:07:00 PM
Age: 18 & Under
Skin: Acne-prone, Tan, Not Sure
Hair: Brunette, Other, Other
Eyes: Brown
This is by far the most disasturous product/system ive tried to help my skin! I followed each step perfectly for about 2 months but stopped because my skin was so dry i could barely smile!! And it didnt help any of my acne go away! The toner seemed to be working but the difference wasnt showing in my skin. The mask didnt work either- it had absolutely no effect! Overall this made my skin worse- and once i stopped using it all together my face broke out like crazy!! I tried the Green Tea moisturizer as well, because i was convinced it would work because of green tea being in the name, but that made me break out! Horrible products in my opinion, i wouldnt reccomend them to anyone
on 7/28/2008 3:41:00 PM
Age: 25-29
Skin: Acne-prone, Medium, Not Sure
Hair: Black, Other, Other
Eyes: Brown
This is the only product that works for me after I've been fighting against the acne for more than 2 years. However, it did dry my skin a little bit, which made it harder to put on foundations.
I just used one set and all my acne is gone.
on 7/27/2008 11:49:00 AM
Age: 18 & Under
Skin: Oily, Medium, Cool
Hair: Black, Straight, Coarse
Eyes: Brown
This is NOT for people with sensitive skin. Trust me, I have extremely annoying oily, very sensitive, acne-prone skin (have had monthly breakouts and blemishes since the end of 6th grade) and ProActiv was my first venture into skin care products. My mom wanted my younger sister and me to try this and - that was our worst mistake ever. Within a few hours, both of our faces were red, acne flaring, it was disaster.
While this may work for some (I have one friend who it's worked decently for), it is NOT for everyone. Give it a shot if you're persuaded by all the celebs they use to promote this products but if you have (or THINK you have) sensitive skin - STAY AWAY! Now I use Cetaphil cleasning gel & moisturizer and Duac & Differin (both prescription) gels. Have helped me with my acne more although I have yet the tackle the problem of my annoying large pores ...
on 7/25/2008 1:38:00 PM
Age: 18 & Under
Skin: Oily, Fair, Not Sure
Hair: Blond, Other, Other
Eyes: Blue
Is this a "miracle" product that has helped millions? Yes. But it didn't work for me. Well, okay, let me clarify.
My skin is oily and very, very acne-prone, and I've tried pretty much every drugstore treatment, prescription-strength otc product, stuff prescribed by various doctors, and nothing has worked.
I've given up on looking for a "quick fix" (after all, my acne has been here for years, I don't hold onto the hope that something will clear it up in a matter of days anymore), and am willing to be patient and consistent while using a system. I used this, and noticed within a day that it was clearing up my skin. (I was using the full three-step system twice daily.) It was getting clearer steadily! Things were looking up! It was working! Until.
One weekend, a spur-of-the-moment camping trip occurred. In the packing frenzy, I forgot my trusty ProActiv. No problem, I thought, I'll only be missing a day or two. I'll just pick back up when we get home!
WRONG. We left Friday around three in the afternoon. By around eight that night, my face started to feel sort of.. acne-y, (it's hard to describe.. sort of an acne premonition). By Saturday morning, there was a fresh crop of tiny pimples! Wow, I thought, that was fast, but whatever. I brushed it off and went about my camping business.
However, it kept coming, and coming, and by Sunday night, my face looked like one of those paintings where you dip your brush in paint and fling it at the canvas, except the paint was the acne and the canvas was my face. It was disgusting, to say the least.
When we got home, I used it, and on Monday morning there was some clearage, and it was pretty much back to where I had started by Wednesday. However, it had started to dry me out (and that's REALLY hard to do! I'm an oil factory!), so my doctor suggested taking it down to just once daily. ProActiv didn't like that (I can't do that, Dave). My acne sprang back up, and by this point I was so pissed about the camping incident, the drying, and the price that I just gave up. This is a miracle product for many, but just not for me.
(However, I have a feeling I'll try it again some day, and how much do you want to bet it'll be a miracle for me too :/ )
on 7/24/2008 3:24:00 PM
Age: 25-29
Skin: Other, Other, Not Sure
Hair: Other, Other, Other
Eyes: Other
I've been using ProActiv 3 step system for 3 years and never been happier! This is THE ONLY product that makes my skin COMPLETELY CLEAR! When you are first starting off using ProActiv, I recommend you to be patient. It took me about 6 weeks for my skin to clear completely.
on 7/24/2008 1:30:00 AM
Age: 18 & Under
Skin: Acne-prone, Medium, Not Sure
Hair: Black, Other, Other
Eyes: Brown
All I have to say is that I'm glad it worked for some, but it made my skin even worse than before. I noticed that there was some clearing up in the beginning stages of using it, but after 1 month of using it I couldn't stand it anymore.
on 7/22/2008 11:10:00 PM
Age: 30-35
Skin: Very Dry, Fair, Not Sure
Hair: Blond, Other, Other
Eyes: Blue
I have occasional acne that seemed ever present when I switched jobs. i tried every Oxy product salicylic acid washing products I could fid. Those things only made my skin WORSE. So I saw the infomercial and decided to try Proactive. This made my skin burn and peel. It realy messed up my skin. I tried it 3 weeks thinking I had to just get used to it or maybe it was renewing my skin cells or something. No, I might as well have dumped BATTERY ACID on my face.
Ladies, stay away from this stuff.
for reference I have pale irish dry skin age 35, no freckles, no blackheads.
on 7/22/2008 2:14:00 PM
Age: 18 & Under
Skin: Acne-prone, Medium, Not Sure
Hair: Blond, Other, Other
Eyes: Green
I fell into the trap that was ProActiv about two years ago, and have been using it continuously ever since, hoping against hope that it would clear up my acne "in time". And it did; off and on. There would be these random times when my face would look better than ever, but then it would just revert back to being a mess for no reason at all. I also found that if I didn't use the toner and the lotion, my face wouldn't be as bad. O.o Weird. I ended up just using the cleanser and that made it a little better.
Anyway! Using this product as a whole did almost nothing for me. Even on my *best* days my acne was still bad. The only thing that was good about this product for me, was the cleanser; the first step. All the other stuff is crap.
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My current project Cosmos, is an examination of experiences involving things not proven by science such as: ghost encounters, clairvoyance and telepathy. It's written that only 4% of the universe is known and we normally use just 10% of our brains. Science admits there is much more going on, but the fallibility of human perception makes research difficult. The human mind is prone to distort reality—even hallucinate emotional realities...and this is a common human practice.
Please join us at 7:30 p.m. on Wed. 24th at Caritas Village for a prayer service honoring the victims of the Charleston tragedy. New Tyler AME church will join with us as we call for peace, forgiveness & healing.
We will be updating you with more info in the next few days!!!
We've got a lot of great entertainment lined up for the main stage. We're excited to announce that Marcella Pinilla will be your emcee for the day! Here's some of the events and entertainment she'll be introducing on stage at Tamale Fest: (Event Schedule)
For the Tamale Fest event map:
(Click here to view and Download map)
Live Entertainment | Tamale Tasting Tent | Tamale Cooking Contest (Pre-Registered Teams Only) | Kids Activity Corner | Food Trucks | Art Vendors | Photo Booth ...and much more
Copyright © 2014 All Rights Reserved By Caritas Village
Caritas Village 2509 Harvard Ave., Memphis, TN 38112 (901) 327-5246
Caritas Village & Caritas Community is a Non-profit 501(c)(3)
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Closed on Sundays and the Holidays of Thanksgiving, Christmas and New Years.


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This Dancing Plumber Is the Best of All Dancing Plumbers
Plumbers are used to getting down and dirty. This plumber, though, is used to just getting down.
Plumbers are used to getting down and dirty. This plumber, though, is used to just getting down.
These turkeys turn the Thanksgiving tables.
Holiday travel is a pain. We all know that. But thank your lucky stars you're not one of these airilne passengers.
Today's "A for effort" story comes courtesy of a determined lil' ferret.
Did you know the pilgrims and Indians shared pickles and salami?
Like pumpkin pie, sweet potatoes and jokes about pardoning turkeys, 'All About That Bass' in inescapable this season.
This guy may be a master chef, but he's got to work on his language skills.
If you've been lamenting the lack of good rowing videos on the Internet, well, today's your lucky day.
Cell phones are as much a part of our world these days as the air we breathe, yet there are still plenty of people who don't know the protocol for using them.
You never know what will happen on the news.
It looks like Pat Sajak may have finally had enough of 'Wheel of Fortune.'
In the annals of bad ideas comes this new offering.


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There's still time to sign up for the annual spring break trip to Washington, D.C. This trip is available to Los Alamos Middle School and home schooled eighth grade students. The trip is a private trip and not a school-sponsored trip. Sign up online at worldstrides.org using ID Number 94821 or call 1-800-468-5899 using the same ID Number.
The trip will include sightseeing in and around Washington, D.C. and Baltimore, Md. Highlights of the trip include the White House, the International Spy Museum, the Walter Reed Medical Museum, a Capitol tour, the Newseum, the Pentagon Memorial, the Washington Monument, the Holocaust Museum, the Smithsonian Museums, the National Zoo, Arlington Cemetery, night tours of the Presidential Monuments, the Iwo Jima, Korean, the Vietnam Memorials, the National Aquarium in Baltimore and much more. The trip will be four days and three nights in duration. The group will stay in a five star hotel in Crystal City, minutes from the D.C. sites.
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Crystal Bennett
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My family history is full of Schmaljohns, Palmers, Hutchisons, Bennetts, Dobbs',Swans,Tiptons,Hillers, the list goes on.
I am also researching the Todd, Grasse, Downey, Greaves families.
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Phil Swan
RE: Samuel Swan
Crystal, this may be your lucky day! I have been hoping for contact from a descendant of Samuel Swan (1835-1885) who was older brother to my grt-grandfather, John Wesley Swan (1838-1913).I have all the information which you are requesting and I am ever so glad to share with you. I do need you to reply to my email address so that I can attach photos, Reports, etc. Please reply to tell me exactly how your are related to Samuel and Mary Ball Swan......a direct lineage from you back to Samuel. You descend from Edward Swann (1630-1707) of Eagleton Plantation. My latest book includes a 450 page Descendant Report on Edward Swann and you will be amazed. I have much information on the siblings of Samuel and John W.
Do yourself a big favor and read my profile.....updated this morning.
best regards,
phil swan email@example.com
Added by
Phil Swan
on Sep 23, 2014 7:45 AM
Carolyn Bierman
Charles McRoy & Baby McRoy
I located and entered the headstone for Charles McRoy. Next to Charles McRoy was a small stone for a Baby McRoy June 23 1928 which I assumed was the Baby Daughter McRoy June 23, 1928 in the Elmwood listing. Since the baby was buried in this plot, I also assumed it was somehow related to the Charles McRoy family. If this is not the correct headstone for that Baby, please let me know and I will delete it. Carolyn
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Carolyn Bierman
on Oct 12, 2013 11:57 AM
Phil Swan
Samuel and Mary Ball Swan
Hi Crystal, I am happy to share all I have on Samuel Swan and other Swans. It is important to me that we communicate via my email firstname.lastname@example.com so that I can send along attachments to you. Before I make a fool of myself, I need to know what you want to know and what you do know. I work with another Swan cousin in MI who is grt-grt granddaughter of Mary Ellen Swan; perhaps you know, I am grt-grandson of John Wesley Swan. We feel we know almost all about the siblings of Samuel and Sue, my MI Swan cousin, has visited the area where Samuel and Mary lived and has researched it well. There is much more that has not and will not get posted on ancestry.com or any other such forum. We have extensive information on our older Swan generations. Enough said here. We're wasting time. If you want my help, I'm willing to help another Swan cousin. Write to me and tell me what you know and what you want to know. I don't need detailed names and dates; I just want to know if you know who are John Swan and Eliz Timms and back from there. Bye for now. Phil Swan
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Phil Swan
on Aug 05, 2013 4:28 PM
John Phillips
I'm not aware of Morris Hill keeping obituaries and I've worked with them a lot. I will add him to my lookup list for the library. It may take me awhile but I will get there within the month to see what I can find for you. Can you email your email address to me so I can send what I find? email@example.com
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on Jul 09, 2013 11:04 AM
Nancy Arendsee
Nona Domke
Hi Crystal,
I'm always excited to find another cousin!! Please contact me at my e-mail address: email@example.com. I would truly appreciate if you could fill me in on Nona's descendants. I will do all I can to help you.
Nancy Arendsee Schaeffer
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on Jul 08, 2013 7:26 AM
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