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The genus Mycobacterium (M.) comprises highly pathogenic bacteria such as M. tuberculosis as well as environmental opportunistic bacteria called non-tuberculous mycobacteria (NTM). While the incidence of tuberculosis is declining in the developed world, infection rates by NTM are increasing. NTM are ubiquitous and have been isolated from soil, natural water sources, tap water, biofilms, aerosols, dust and sawdust. Lung infections as well as lymphadenitis are most often caused by M. avium subsp. hominissuis (MAH), which is considered to be among the clinically most important NTM. Only few virulence genes from M. avium have been defined among other things due to difficulties in generating M. avium mutants. More efforts in developing new methods for mutagenesis of M. avium and identification of virulence-associated genes are therefore needed.
We developed a random mutagenesis method based on illegitimate recombination and integration of a Hygromycin-resistance marker. Screening for mutations possibly affecting virulence was performed by monitoring of pH resistance, colony morphology, cytokine induction in infected macrophages and intracellular persistence. Out of 50 randomly chosen Hygromycin-resistant colonies, four revealed to be affected in virulence-related traits. The mutated genes were MAV_4334 (nitroreductase family protein), MAV_5106 (phosphoenolpyruvate carboxykinase), MAV_1778 (GTP-binding protein LepA) and MAV_3128 (lysyl-tRNA synthetase LysS).
We established a random mutagenesis method for MAH that can be easily carried out and combined it with a set of phenotypic screening methods for the identification of virulence-associated mutants. By this method, four new MAH genes were identified that may be involved in virulence.
Keywords:Mycobacterium; Mycobacterium avium subsp. hominissuis; Non-tuberculous mycobacteria; Virulence; Mutagenesis; Illegitimate recombination
The genus Mycobacterium (M.) comprises highly pathogenic bacteria such as M. tuberculosis as well as environmental opportunistic bacteria called NTM. They are ubiquitous and have been isolated from soil, natural water sources, tap water, biofilms, aerosols, dust and sawdust [1-3]. Remarkably, NTM are resistant to amoeba and protected against adverse conditions inside amoebal cysts [4]. While the incidence of tuberculosis is declining in the developed world, infection rates by NTM are increasing [5]. NTM cause skin infections, lung diseases, lymphadenitis and disseminated disease mostly in immuno-compromised persons [5]. Lung infections as well as lymphadenitis are most often caused by M. avium[5,6], and M. avium is considered to be among the clinically most important NTM [7].
M. avium can be divided into four subspecies. M. avium subsp. paratuberculosis (MAP) causes the Johne's disease in ruminants; M. avium subsp. avium (MAA) and M. avium subsp. silvaticum infect birds; and finally M. avium subsp. hominissuis (MAH) which causes disease in humans [8].
The main route of infection in AIDS patients is the invasion of mucosal epithelial cells of the gastrointestinal tract, while in non-AIDS patients infections mainly occur through the respiratory route [9]. Recognition of M. avium by mouse macrophages involves binding of a 20 – 25 kDa lipoprotein from the cell envelope of M. avium to TLR2. This interaction leads to bacteriostasis of M. avium in a MyD88-dependent way [10]. Even though the expression of TNF-α is also induced via TLR2-signalling, its role in growth restriction of M. avium is unclear [10]. IFN-γ is considered to be a key cytokine for killing of M. avium and its expression is promoted by IL-18 secreted by M. avium-infected human macrophages [11]. Phagocytosis of M. avium is supposed to be mediated via binding of the bacteria to a variety of receptors including complement receptors CR1, CR2, CR3, CR4, the mannosyl-fucosyl-receptor, the fibronectin receptor, the integrin receptor α(v)β3, and the transferrin receptor [12-15]. M. avium inhibits the acidification of the phagosome and the fusion of the phagosome with lysosomes [16,17].
Intracellular M. avium survives antibacterial activities such as nitric oxide and reactive oxygen species and the mechanisms leading to killing of M. avium are still unknown [18]. The cell wall structure is an important factor determining virulence of M. avium[19]. Thus, different colony morphotypes (smooth opaque, smooth transparent, rough) distinguishable on Congo Red plates display different degrees of virulence. Smooth transparent and rough colonies are considered to be more virulent than smooth opaque colonies [20,21]. The colony morphotype is associated with the glycopeptidolipid (GPL) composition [19]. By inducing the release of various pro-inflammatory cytokines such as IL-1, IL-6 or TNF-α, GPL modulate the immune response against mycobacteria [22].
Only relatively few virulence genes from MAH have been defined with respect to their role in infection. This is partly attributable to difficulties in generating MAH mutants. The major obstacle is the low transformation frequency if MAH is used as recipient. This also limits the efficiency of so far described random mutagenesis systems, such as the commercially available EZ-TN < KAN2 > Tnp Transposome from Epicentre. This Tn903-based system consists of a stable complex formed between the EZ::TN Transposase enzyme and the EZ::TN < KAN-2 > Transposon. It was used in MAA and MAH to analyse mechanisms of multidrug resistance and the role of GPL [23-25]. Another system for the generation of random mutants is based on transduction using temperature-sensitive phages containing a transposon with a selection marker [26,27]. In other mycobacterial species such as M. tuberculosis and M. bovis BCG linear recombination substrates have been applied to generate random as well as site-directed mutants [28-30]. This approach, however, so far has not been published for mutagenesis of MAH or MAA. With the present study we intended to explore the performance of illegitimate recombination of a linear recombination substrate for random mutagenesis of MAH.
Bacterial strains, amoeba, cell lines and growth conditions
Mycobacterial strains were grown in Middlebrook (MB) 7H9 broth (BD Biosciences, USA), supplemented with either 10% ADC (BD Biosciences) or 10% OADC (BD Biosciences) and 0.05% Tween 80 without shaking, and on MB 7H11 agar (BD Biosciences) at 37°C. Escherichia coli DH5α was used as host strain for plasmid pYUB854, a cosmid vector with a Hygromycin resistance (Hygr) gene [31] and was cultured in/on Luria-Bertani broth and agar at 37°C. Antibiotics when required were added at the following concentrations: Kanamycin (50 μg ml-1) or Hygromycin (50 μg ml-1). For Congo Red plating agar media was supplemented with 100 μg ml-1 Congo Red. The Acanthamoeba castellanii strain 1BU group II [32] was cultivated in PYG medium (Proteose peptone-Yeast extract-Glucose [33]) at 28°C and passaged once per week. The human macrophage cell line THP-1 (DSMZ-No. ACC-16, DSMZ GmbH, Braunschweig, Germany) was maintained by passaging twice weekly in RPMI 1640 (GIBCO® Invitrogen, Darmstadt, Germany) supplemented with 10% foetal bovine serum (Bio Whittaker, Walkersville, MD, USA). Cells were cultured in BD FalconTM 75 cm2 trays (BD Biosciences) at 37°C and in 5% CO2. For human macrophages infection and washing, Iscove's Modified Dulbecco's Media (IMDM) (PAA laboratories, Austria) with 3% Human AB-serum (PAA laboratories) was used.
Molecular biology techniques
All molecular biology techniques were carried out according to standard protocols [34] or according to the recommendations of the manufacturers of kits and enzymes. Primers were purchased from Metabion (Martinsried, Germany). Plasmid DNA was isolated with the QIAGEN Plasmid Mini Prep Kit (Qiagen, Hilden, Germany). Polymerase chain reaction (PCR) was performed with the DreamTaq Kit from Fermentas (St. Leon-Rot, Germany). Restriction enzymes were purchased from Fermentas. For elution of DNA fragments from agarose gels, the QIAquick Gel Extraction kit (Qiagen) was used. Ligation reactions were performed with the T4 DNA Ligase Kit from Fermentas. Genomic DNA from mycobacteria was isolated according to the protocol described in Sjöbring et al.[35]. Sequencing reactions were performed by using the Prism Big Dye FS Terminator Cycle Sequencing Ready Reaction Kit from PE Applied Biosystems (Darmstadt, Germany). Nucleotide sequence analysis was performed using the software packages MacVector™ 7.2.3 (Accelrys, Cambridge, UK) and Lasergene (DNASTAR, Inc., Madison, WI, USA).
For Southern blotting 2 μg of genomic DNA from Mycobacterium were digested with ApaI or SmaI, separated by electrophoresis in a 1% agarose gel and capillary transferred to positively charged nylon membranes (GE Healthcare, Buckinghamshire, UK) by following a standard protocol [34]. An 1818 bp region of the plasmid pYUB854 carrying the Hygr gene was amplified using the primer pair Hyg2K FW (5´-CAC CGT ACG TCT CGA GGA ATT CCT G-3´) and Hyg2K BW (5´-GCG TCG TGA AGA AGG TGT TGC TGA-3´) and the digoxigenin labeling Kit (Roche, Mannheim Germany). The labeled PCR-product was used as a probe and detection was carried out using anti-digoxigenin-AP conjugate and CDP-star (Roche) according to the manufacturers' instructions.
Reverse PCR was applied to exactly locate the insertion sites of the Hygr gene in the mutants. 2 μg of DNA of each mutant was digested with the restriction enzyme ApaI or SmaI (which do not cut in the recombination substrate). The multiple sized DNA fragments were ethanol precipitated and then self-ligated by T4 DNA ligase enzyme, thus resulting in different sized circular DNA molecules. A PCR was then performed with primers [Hyg mut_1 (5´-AAC TGG CGC AGT TCC TCT G-3´) and Hyg mut_2 (5´-TCA GCA ACA CCT TCT TCA CGA-3´)] binding within the Hygr gene and oriented towards the unknown genomic MAH DNA located adjacent to the Hgyr gene. Sequencing of the PCR products using the primers Hyg mut_1 and Hyg mut_2 followed by BLAST analysis of the sequences allowed the exact identification of the insertion sites of the recombination substrates.
For quantitative RT-PCR the mutants were grown in MB/ADC with 25 μg ml-1 of Hygromycin B to an OD600 of 2. The pellet of 10 ml of culture was resuspended in 4 ml of protoplasting buffer (15 mM of Tris–HCl pH 8, 0.45 M of Sucrose, 8 mM of EDTA) with 4 mg ml-1 Lysozyme. After incubation at 37°C for 45 minutes (min) the protoplasts were harvested by centrifugation and the pellets were resuspended in 1050 μl of the RLT buffer from the RNeasy Minikit (Qiagen) with 10.5 μl of ß-Mercaptoethanol. This suspension was transferred into tubes containing 25–50 mg of glass beads (0.5 mm, PeqLab, Erlangen, Germany) and shaken in the homogenizer Precellys 24 (PeqLab) for 45 sec at 6,500 g. The tubes were chilled on ice and centrifuged at 8,000 g for 5 min at 4°C. Then, 0.7 volume of absolute Ethanol was added to the supernatant and this solution was distributed onto two columns of the RNeasy Kit. The samples were further processed as described in the RNeasy manual. Residual DNA present in the RNA preparations was removed with the Kit Desoxyribonuclease I (DNaseI) RNase free from Fermentas. The M-MLV Reverse Transcriptase and Random primers from Promega (WI, USA) were used to transcribe cDNA from the RNA. The cDNA was then used to perform real time PCR with the MaximaTM SYBR Green/Rox qPCR Master Mix 2x from Fermentas. Primers were (i) for gene MAV_1779: 5´-CTG CAG AAG AGC GTC TAC CC-3´ and 5´-CTC TGT TCG GAG GTC GTC AT-3´, (ii) for gene MAV_3129: 5´-GGT CAA GAC CAT CGA CGA CT-3´ and 5´-AGA TCA TGA ACG GCA CAA CA-3´, (iii) for gene MAV_4332: 5´-ATG GTC GAG CAG AGC ATC TGG-3´ and 5´-ATG GCG TCC ACG AAC CG-3´, (iv) for gene MAV_5105: 5´-GTT GTC GAG TTC ACC GGT CT-3´ and 5´-ATT CAC TCG GCG AAT ACC TG-3´ and (v) for 16S rRNA gene: 5´-GAG TGA GAA TGC AGG CAT GA-3´ and 5´-ACA CGG GTA CGG GAA TAT CA-3´. The ΔΔCT method was used to calculate the relative expression of the gene of interest in the mutant in comparison to the mean of its expression in the other three mutants. Normalisation was obtained by measuring the expression of 16S rRNA gene as reference gene.
Random mutagenesis by illegitimate recombination
1 μg of plasmid pYUB854 DNA was double digested with restriction enzymes StuI and SpeI Fast digest at 37°C for 30 min. The 2030 bp linear DNA fragment carrying the Hygr gene was gel-eluted after electrophoresis and 3–6 μg linear DNA fragment was transformed into M. avium strains by electroporation with the Biorad GenePulser apparatus applying 1000 Ω, 25 μF and 1.25 kV in 1 mm gap cuvettes. The preparation of electrocompetent cells and electroporation were performed using standard protocols [36]. Plasmid pMN437 was used as positive control for transformation [37]. Electroporated bacteria were incubated at 37°C for 24 hours (h) before plating on selective plates. Potential mutants were characterised by PCR amplifying a part of the Hygr gene [primers Hyg 2 K LC FW (5´-AGT TCC TCC GGA TCG GTG AA-3´) and Hyg 2 K LC BW (5´-AGG TCG TCC CGG AAC TGC TGC G-3´)], Southern blotting, reverse PCR (primers Hyg mut 1 and Hyg mut 2) and sequencing.
Construction of a complemented derivative of mutant MAV_3128
Primers MAV3128_MV306_1 (5´-CGG
Screening for virulence-mutants
Amoeba Plate Test (APT)
The APT was previously described [40]. In short, known concentrations of Acanthamoeba castellanii (1BU group II strain) diluted in PYG medium were spread on MB agar plates and these plates served as test plates. For control plates only PYG medium without amoeba was spread on MB agar plates. Plates were dried and incubated at room temperature. The next day series of tenfold dilution (1:10, 1:100, and 1:1000) in sterile water were prepared from cultures of the mutants and the M. avium 104 wild type (WT). 3 μl of undiluted culture and of each dilution were spotted onto the test and control agar plates. Plates were then incubated at 30°C for one week. Mutants showing reduced growth on test plates compared to the control plates were selected for further molecular characterisation.
Growth rate in broth cultures under pH stress
The growth rates of mutants and WT were compared in MB 7H9 broth with neutral pH (7) and under pH stress (pH 5). Cultures were inoculated to an initial OD600 of 0.02 to 0.03 and allowed to grow for two weeks. Three cultures per strain were inoculated. Growth of cultures was determined by measurement of OD600 of cultures and also by quantification of ATP with the luminescence-based Kit BacTiter-GloTM Microbial Cell Viability Assay (Promega). The luminescence was recorded as relative light units (RLU) with the microplate luminometer LB96V (EG & G Berthold). Mutants showing differences of growth pattern compared to the WT in both neutral medium and under pH stress conditions were considered for further molecular characterisation.
Congo Red plating
100 μl of 1:105 and 1:106 dilutions in sterile water of mutants, complemented strain and WT were spread in triplicate on MB agar plates supplemented with OADC and 100 μg ml-1 Congo Red. Plates were incubated for 2–3 weeks and observed for colony morphology. Mutants showing differences in colony morphology (white vs. red staining, transparent vs. opaque colonies, smooth vs. rough colonies) compared to the WT were considered for further molecular characterisation.
Induction of cytokine expression in THP-1 cells
Infection of the cell line THP-1 was performed in 24-well cell culture plates (TPP) with three to five wells per sample. A total of 200,000 cells per well of THP-1 were grown along with addition of phorbol-12-myristate-13-acetate (PMA, Sigma, Taufkirchen, Germany) (10 ng ml-1) and allowed to adhere to the surface of the plate well overnight at 37°C and in 5% CO2. Cells were then infected with mutants and WT at a multiplicity of infection (MOI) of 50 colony forming units (CFU). The supernatants were removed after 24 h and cytokines were quantified in appropriate dilutions of the supernatants by ELISA using the Human ELISA Ready to go Kits (Natutec, Frankfurt, Germany).
Intracellular survival in THP-1 cells
THP-1 cells were seeded, treated with PMA and infected as described above. The supernatants were removed after 4 h infection period and adherent cells were washed twice with RPMI 1640. The cells were then treated with 200 μg ml-1 of Amikacin (Sigma) for 2 h to kill the mycobacteria in the supernatant. After washing twice with PBS buffer (10 mM sodium phosphate, 126 mM sodium chloride, pH 7.2), 1 ml of medium supplemented with 5 μg ml-1 of Amikacin was added to each well. Samples for quantification of intracellular bacteria were taken at the end of the infection time after removal and killing of extracellular bacteria and then after 1, 2, and 4 days. For this, the cells were lysed in 1 ml of water at 37°C for 20 min and the mycobacterial DNA in the lysates was quantified by real-time PCR as described in Lewin et al.[41]. Additionally, 100 μl of 1:103 dilution in sterile water of samples were plated in triplicate on agar plates supplemented with ADC for counting of CFU.
Intracellular survival in human monocytes
Human monocytes were isolated from buffy coats from healthy donors using Ficoll-PaqueTM Plus (GE Healthcare) and PercollTM (GE Healthcare) gradient centrifugation according to the manufacturer's recommendations and as described in Sharbati et al.[42]. One million macrophages were seeded per well in 24-well cell culture plates, with three to five wells per sample per sampling point. Infection with mutants, complemented strain and WT, Amikacin treatment and sampling were done as described above for THP-1 cells infection, except that human monocytes were pre-activated with 100 U ml-1 of human IFN-γ (Invitrogen, Darmstadt, Germany) and 10 ng ml-1 of LPS (Sigma), IMDM was used for washing, the MOI for infection was 10 and the dilution of the samples for plating and counting of CFU was 1:500.
Results and discussion
Generation and genetic characterisation of M. avium mutants
Our aims were the establishment of a new method to mutagenise MAH and the identification of mutants potentially affected in virulence. The mutagenesis approach involved transformation of a recombination substrate by electroporation into MAH, and we therefore first identified clinical and environmental MAH strains applicable to electroporation. We considered a prior investigation of transformability to be necessary, because other authors had reported some clinical M. avium strains to be inaccessible to electroporation [43]. As proposed by Lee et al.[43], we chose a gfp-containing plasmid (pGFP: gfp cloned in vector pMV261 [38]) for transformation assays. We tested 14 clinical isolates and two soil isolates. Strain M. avium 104 was originally isolated from an HIV patient [44] and strains 2721/04, 10091/06, 10203/06, 4557/08, 4023/08, 3646/08, 3449/08, 3269/08, 2630/08, 2014/08, 772/08, 709/08, 528/08 were isolated from children with lymphadenitis. Strains 128 and 129 are soil isolates. Out of these 16 M. avium strains, five (104, 2721/04, 2014/08, 4023/08 and 528/08) could be transformed with pGFP. As the genome sequence from M. avium strain 104 is available in the genome data bases, simplifying a precise mutant description, we decided to concentrate on this strain for further analysis.
Our mutagenesis approach took advantage of the high rate of illegitimate recombination in slow growing mycobacteria [28,45] and their ability to take up linear DNA [29]. For selection purposes we chose the Hygr gene instead of also often used Kanamycin resistance gene (Kmr), because the Hygr gene had been shown before to be superior to the Kmr gene especially for the transformation of other than laboratory strains [46]. The Hygr gene used for electroporation was flanked by plasmid DNA of 793 bp on one side and 238 bp on the other side. These flanking regions served as substrates for the illegitimate recombination. After electroporation of 3–6 μg of restriction fragment and selection on plates containing Hygromycin, about 1000 colonies could be obtained. Around ten percent of this number appeared on the negative control plates (no DNA added) and had to be considered spontaneously resistant. The positive controls (with 1–2 μg plasmid DNA) generated around 5–6 times more colonies than could be observed on the test plates. Transposon/transduction mutagenesis procedures have been reported to deliver around 1,000 to 3,500 mutants per mutagenesis procedure [19,23,24,27,47,48] which means that the efficiency or our method was below the efficiency of transposon/transduction systems. Taking into account the simple handling of our method we consider it nevertheless to be a good alternative to the currently applied methods for mutagenesis of MAH.
Fifty randomly chosen colonies from the sample plates were tested for insertion of the Hygr gene by performing a PCR using the primers Hyg 2 K LC FW and Hyg 2 K LC BW (data not shown). By this PCR 49 of the 50 colonies could be confirmed to carry an insertion of the Hygr gene in the genome. Additionally, Southern blots using a PCR fragment produced with primer pair Hyg2K FW and BW as probe were performed to verify if the insertions had occurred at different genome sites in different colonies (data not shown). Hybridising bands were obtained with the DNA from 20 colonies and confirmed independent insertion events.
Inverse-PCR using the primers Hyg mut 1 and Hyg mut 2 followed by sequencing of the PCR products enabled us to identify the sites of insertion of the Hygr gene in 13 mutants. As shown in Figure 1, there were no hot spots for integration but the insertions were distributed within the whole M. avium genome.
Figure 1. Sketch showing randomly mutated genes distributed within the M. avium genome. Genes location mapped on the genome after sequencing.
The genetic characterisation of four virulence-associated mutants is shown in Figure 2. The integration events were accompanied by deletions in all 13 mutants. The smallest deletion had a size of 2 bp, the largest one of 669 bp. All insertions were located within coding regions. Only in one mutant more than one gene was affected by the insertion. In 12 of the 13 mutants the linear recombination substrate had been completely inserted and in one mutant the inserted fragment had been shortened at both ends. The sequences next to the inserted fragment showed no special structure or nucleotide sequences.
Figure 2. Sketch illustrating the genetic characterisation of the mutants MAV_1778, MAV_3128, MAV_4334, and MAV_5106. The sites of the insertion of the marker (Hygr gene) were identified by inverse PCR followed by sequencing of the eluted PCR products. The figure shows for four mutants the mutated gene (dark blue) with the site of insertion of the fragment (grey) carrying the Hygr gene (red) and the four genes located upstream and downstream of the mutated gene (light blue). Numbers in the arrows indicate the gene names. The direction of the arrows stands for gene direction. Gene sizes and distances between genes are approximations. Below the map of each mutant the size of the deletion generated as result of insertion of the marker is indicated.
Both, the random distribution of insertion sites and the low rate of large deletions affecting more than one gene are benefits of our method. Contrary to our experience with MAH, Collins and colleagues [49] observed more clustered insertions and deletions of up to 12 genes by mutagenising M. bovis with a DNA fragment carrying a Kanamycin resistance gene by illegitimate recombination. It would be interesting to find out the reasons for these differing outcomes. Are the specific parameters of the illegitimate recombination events species-specific or does the composition of the recombination substrate play a more important role?
In favor of a straight forward procedure, we concentrated our further efforts on those mutants, which fulfilled the following requirements: - an insertion in the middle of the coding region of a gene, – mutation of only one gene and - mutation of a single copy gene. After applying these criteria, eight mutants (see Table 1 for mutated genes and their functions) were selected for phenotypic analysis.
Table 1. Mutated M. avium genes and their functions
Phenotypic characterisation of MAH mutants
Since virulence is regulated on many different levels we applied more than one screening test (as for example intracellular multiplication) to identify a greater spectrum of relevant virulence-associated genes. We searched for phenotypic assays allowing a fast screening of our mutants and not requiring special and expensive equipment. The selected tests should monitor changes in (i) cell wall composition (plating on Congo Red Agar), (ii) resistance towards low pH, (iii) amoeba resistance, (iv) induction of cytokine secretion by infected macrophages and (v) intracellular survival and growth in human macrophages.
Colony morphology and Congo Red staining characteristics
The occurrence of different colony morphotypes is an eye-catching feature of M. avium including MAH and has attracted attention also because it is associated to virulence [19,24,50,51]. The colony morphology is influenced by the composition of the cell wall, which is a major determinant of mycobacterial virulence [52-54]. Congo Red, a planar hydrophobic molecule can bind to diverse lipids and lipoproteins and is thus applicable for the detection of changes in cell wall composition [54-56]. Upon plating of MAH on Congo Red agar plates, smooth transparent, smooth opaque and rough colonies as well as red and white colonies can be distinguished. Appearance of the three morphotypes smooth opaque, smooth transparent and rough was also confirmed for strain 104 [21]. While the opaque-transparent switch is reversible, the rough phenotype results from irreversible deletion of cell envelope glycopeptidolipid genes and is irreversible [24,51].
TLC (Thin Layer Chromatography) analysis of the different morphotypes from strain 104 has been performed by Torelles [21]. They also analysed the sugar composition of the glycopeptidolipids (GPL) by gas chromatography–mass spectrometry (GC–MS) analysis. They found that the smooth opaque and smooth transparent colonies formed similar GPL and both expressed besides the nsGPL (ns: non-specific) the ssGPL (ss:serovar specific) of serovar 1. However, the ssGPL was absent in the rough morphotype, which had a strong band of the nsGPL. A band in the lipopeptid region devoid of sugars was present in the smooth transparent morphotype and the rough morphotype but lacking in the smooth opaque morphotype. The sugar composition of all morphotypes showed the typical profiles related to ns and ssGPL of serovar 1, only in the rough morphotype 6-deoxytalose and 3-O-methyl-6-deoxytalose were missing.
The transparent colony variant grows better in macrophages and animals compared to the opaque variant. Moreover, white transparent colonies survived better in macrophages than red transparent colonies [19,24,50,51,56]. These differences in intracellular survival may be caused by variations in the cytokine response towards infection by different morphotypes. The smooth opaque morphotype has been shown to induce higher levels of secretion of IL-1α, IL-1β and TNF-α by human blood-derived monocytes compared to the smooth-transparent morphotype [57]. Variation in cytokine response upon infection with either smooth-opaque or smooth-transparent M. avium was also reported upon infection of human microglia cultures [58].
The colony morphology of the WT and the mutants upon plating on Congo Red Agar is shown in Figure 3. The WT (Figure 3 A) mainly formed smooth-domed-opaque (sdo) colonies along with smooth-transparent (st) colonies. Mutant MAV_2555 showed the same morphologies, but additionally smooth-flat-red (sfr) colonies were visible (Figure 3 B). Relatively few smooth-transparent and rough colonies occurred in mutant MAV_1888 (Figure 3 C), MAV_4334 (Figure 3 D) and MAV_5106 (Figure 3 E). Mutant MAV_4334 (Figure 3 D) showed a higher variation with respect to the intensity of red color of smooth-domed-opaque colonies. Mutant MAV_1778 showed a very high degree of variability displaying red-rough (rr) and smooth-flat-red colonies additionally to the smooth-domed-opaque, smooth-transparent and rough-white (rw) colonies (Figure 3 F). The colonies generated by mutant MAV_3128 (Figure 3 G) were in average larger in size and the smooth-opaque colonies appeared paler than in the WT. Also, the edges of these colonies were more irregular. Some red-rough colonies were also visible. The most multifaceted image was displayed by mutant MAV_3625. This strain generated smooth-domed-opaque, smooth-domed-red (sdr), smooth-flat-red, smooth-transparent and rough-transparent (rt) and red-rough colonies (Figure 3 H). A high proportion of red colonies (smooth-domed-red, smooth-flat-red, red-rough) was generated by mutant MAV_2599 (Figure 3 I) additionally to smooth-opaque and smooth-transparent colonies. This mutant produced only few rough (rough-transparent, rough-red) colonies. Altogether, we observed a high frequency and intensity of morphological changes in the mutants pointing to involvement of the mutated genes in the composition of cell wall structure. Since studies by different authors have related colony morphotype to virulence it would be of interest to investigate in further experiments if and to which degree the different colony types are stable and differ in their virulence.
Figure 3. Colony morphology upon plating on Congo Red agar plates. Well-grown broth cultures of all strains were diluted 1:106 and 100 μl plated in triplicate onto Middlebrook agar with OADC containing 100 μg ml-1 Congo Red. Plates were incubated on average for three weeks. The arrows point to smooth-domed-opaque (sdo), smooth-flat-red (sfr), smooth transparent (st), rough red (rr) and rough transparent (rt) colonies. A: WT; B: mutant MAV_2555; C: mutant MAV_1888; D: mutant MAV_4334; E: mutant MAV_5106; F: mutant MAV_1778; G: mutant MAV_3128; H: mutant MAV_3625; I: mutant MAV_2599.
The intraphagosomal pH of M. avium-containing phagosomes decreases to pH 5.2 in activated macrophages [59]. We therefore investigated the pH-resistance of the mutants compared to the WT by inoculating them into MB broth at pH 5 and pH 7 and measuring the growth during 11 days at 37°C by means of OD measurement and ATP quantification. ATP measurement represents a much more sensitive method than the OD measurement. Additionally, the OD of a culture not only depends on cell number but also on the size of the cells, their morphology and the degree of clumping of the cells. For these reasons, ATP measurement was reported to be a more reliable method for quantification of mycobacteria in broth culture [41]. As shown in Figure 4, the WT grew better at neutral pH than at low pH. After 11 days of growth in neutral medium, it generated 722,491 RLU (relative light units), while in medium with acidic pH only 143,082 RLU were achieved. The mutants MAV_2555, MAV_1888, MAV_4334 and MAV_5106 showed a similar growth pattern as the WT, both in neutral and acidic pH (data not shown). The mutants MAV_1778 and MAV_3128 grew similar as the WT at neutral pH; however, at low pH these strains enhanced their growth rate even above the level reached at neutral pH (Figure 4 A and B). While the mutant MAV_3128 showed enhanced growth in comparison to the WT at low pH already at day 1, the mutant MAV_1778 showed an identical growth rate as the WT at low pH until day 5 and then showed strongly enhanced growth. The mutants MAV_3625 and MAV2599 grew better than the WT at pH 7 and were able to maintain this growth rate at pH 5 (Figure 4 C and D). In summary, the mutations either had no influence on the survival under pH stress conditions or improved resistance towards pH stress.
Figure 4. Resistance towards pH stress. The bacteria were grown in Middlebrook 7H9 broth with OADC at pH 7 and pH 5 during 11 days; the ATP content was recorded by quantification of the amount of ATP in the cultures. The amount of ATP is represented as RLU (relative light units). A: WT and mutant MAV_1778; B: WT and mutant MAV_3128; C: WT and mutant MAV_3625; D: WT and mutant MAV_2599.
Amoeba plating test
Free-living amoebae are known to host environmental mycobacteria including M. avium, which are able to survive in Acanthamoeba trophozoites as well as in the exocysts [4,60,61]. Growth in Acanthamoeba was associated with subsequently enhanced virulence in infection experiments with mice [62]. Since some virulence mechanisms are employed by amoeba-resistant bacteria to survive in amoebae as well as in macrophages [4,63-65], amoebae have been used as test systems for determination of bacterial virulence factors [40,63,66]. An Acanthamoeba castellanii agar plate assay was developed and successfully employed for screening of mutants of Legionella pneumophila[40]. We adapted this APT to fit the growth conditions (medium, temperature, duration) of M. avium and tested the eight mutants in comparison to the WT. After incubation for five to seven days at 28°C, the WT formed colonies even if the cultures were diluted 1:103 before being dropped on the lawn of amoebae. The growth of some mutants was more strongly affected by the amoebae but a differentiated evaluation of the impact of the various mutations on survival in the amoebae was not possible (data not shown). The APT thus was not sensitive enough to reveal differences in the capacity of the mutants to survive within the amoebae. This was surprising, because the APT has proven to be an efficient tool for the identification of virulence genes in L. pneumophilae[40]. There are several possible explanations for this discrepancy. Amoebae are the most important habitat of Legionella, while M. avium is not dependent on the presence of amoebae for survival and distribution. As a consequence, Legionella might have evolved more important virulence factors interacting with amoebae. Another possible explanation may result from the differences in the generation times of L. pneumophilae and M. avium. L. pneumophilae is a fast-growing bacterium forming clearly visible colonies few days after plating, while the slow-growing M. avium 104 requires two weeks to generate colonies of comparable size. This time span may be too long to maintain the amoebae as trophozoites actively interacting with the mycobacteria. In conclusion, we estimate the APT to be of only little value for the detection of virulence genes of slow-growing mycobacteria.
Induction of cytokine secretion
The innate immune recognition by phagocytic cells mediates cellular activation enabling killing of the bacteria and the production of pro- and anti-inflammatory cytokines. The signaling cascade is mainly initiated by binding of M. avium components to TLR2 followed by recruitment of the MyD88 adaptor molecule and the activation of NFκB and MAP kinases. This chain of events ends with the induction of inflammatory cytokines [10] controlling macrophage activation and granuloma formation. We monitored the induction of cytokine expression of THP-1 macrophages by the WT and the mutants in order to evaluate their ability to stimulate the immune signaling. To this aim we quantified the secretion of selected cytokines: the pro-inflammatory cytokines TNF-α, IL-1β and the anti-inflammatory cytokine IL-10. Five independent experiments were normalised for WT (expression ratio 1) to determine the expression ratio for the mutants in comparison to WT. While results for TNF-α and IL-1β were not significantly different as compared to WT, IL-10 was significantly (P <0.007) up-regulated for mutant MAV_4334 (Figure 5). IL-10 can inhibit the production of inflammatory cytokines such as TNF-α in monocytes pre-activated by IFN-γ and LPS [67,68] and therefore plays an important role in the immune response.
Figure 5. Induction of IL-10 cytokine secretion by infected macrophages. THP-1 cells (2.0x105) were infected (MOI 50) with mutants and WT. After 24 hours cytokines from supernatants were measured by ELISA. When compared to WT a P value <0.01 (two-tailed, unpaired Mann–Whitney test) was considered very significant (**).
Intracellular survival
The ability to survive and even replicate inside the phagosomes of macrophages is an important virulence factor of mycobacteria and was therefore included in our screening options. Infection experiments with macrophages give information on the early host response to mycobacterial infections [69]. Different types of macrophages or monocytic cells have been employed to assess mycobacterial virulence and among these the human macrophage-like cell line THP-1 has proven a suitable system for virulence testing [69,70]. It was shown that THP-1 cells are similar to primary human monocyte-derived macrophages with respect to their ability to take up mycobacteria and limit their growth [71]. We infected THP-1 cells that had been differentiated by PMA with the WT and the mutants. Intracellular mycobacteria were measured by quantitative real-time PCR and CFU by plating. Survival of mutants in THP-1 cells was not consistently different if compared to the WT (data not shown). More significant differences were obtained when using human blood monocytes for the infection experiments. The growth of mutant MAV_4334, MAV_1778 and MAV_3128 was affected the most in human monocytes (Figure 6). They were reduced significantly for the first two days (P < 0.05 to P < 0.01). Mutant MAV_4334 and MAV_1778 (Figure 6 A and C) were almost reduced to half during the first two days. As shown in Figure 6 D, mutant MAV_3128 had the highest significant (P < 0.001) difference in growth as compared to WT, which had survived better during this time period. The mutant MAV_5106 largely differed from other mutants and during four days of infection had shown constant survival (Figure 6 B). The capacity of mutant MAV_5106 to survive better in macrophages suggests that it may be characterised by a higher virulence as compared to the other mutants. Tateish et al.[70] compared the virulence of different M. avium isolates in humans, immuno-competent mice and THP-1 cells. They found that the strain causing the most serious disease in humans and the highest bacterial load in mouse lungs also grew better in THP-1 cells than the other strains tested. According to this, the mutants MAV_4334, MAV_1778 and MAV_3128 may display reduced virulence and the corresponding genes may represent virulence-associated genes.
Figure 6. Intracellular survival of mutants compared to WT in human monocytes. Human blood monocytes (1.0x106) from healthy volunteers were infected (MOI 10) with mutants and WT. Intracellular bacteria were quantified after 4 hour of infection, and after 1, 2, & 4 days. The monocytes were lysed in 1 ml of sterile water and 100 μl of 1:500 dilution in sterile water of sample were plated on Middlebrook agar plates supplemented with ADC for CFU counting. A: WT and mutant MAV_4334; B: WT and mutant MAV_5106; C: WT and mutant MAV_1778; D: WT and mutant MAV_3128. Statistical analysis was done using a two tailed, paired Student's t test. When compared to wild-type a P < 0.05 was considered significant (*) and a P < 0.01 very significant (**).
Evaluation of the screening procedure
We have employed five screening methods (colony morphology, pH stress resistance, amoeba resistance, cytokine induction, intracellular survival) to select mutants affected in virulence-related traits. Two mutants (MAV_4334 and MAV_3128) responded differently from the WT in four of these five screening tests and two mutants (MAV_5106 and MAV_1778) reacted differently in three screening tests. The most prominent differences were exhibited by mutant MAV_3128. The other mutants either did not show any differences compared to the WT or reacted differently in only one or two tests.
The insertions in mutants MAV_4334, MAV_5106, MAV_1778 and MAV_3128 have been mapped and the structure of the mutated regions has been analyzed on nucleotide level. In all cases only one gene has been mutagenised. The insertions are located in the genes MAV_4334 (nitrogenase reductase family), MAV_5106 (phosphoenolpyruvate carboxykinase), MAV_1778 (GTP-binding protein LepA) and MAV_3128 (lysyl-tRNA synthestase LysS).
Phosphoenolpyruvate carboxykinases (PEPCK) catalyse the reversible decarboxylation and phosphorylation of oxaloacetate to form phosphoenolpyruvate. Mutations of the PEPCK gene from M. bovis BCG are characterised by attenuated virulence and reduced survival in macrophages [72]. The PEPCK gene from M. tuberculosis was shown to be required for replication in murine bone marrow macrophages and mice [73].
The LepA protein from M. tuberculosis possess GTPase activity. Bacterial GTP-binding proteins play a role in regulation of ribosomal function and cell cycle, modulation of DNA partitioning and DNA segregation [74]. In Helicobacter pylori LepA is important for growth at low pH and may play a role in infection [75].
The lysS gene from M. avium is 81% homologous to the lysX gene from M. tuberculosis. LysX from M. tuberculosis is required for synthesis of lysinylated phosphatidylglycerol. A LysX mutant was shown to be sensitive to cationic antibiotics and peptides, to be more lysosome-associated and to display defective growth in mouse and guinea pig lungs [76].
So far, nothing is known about the role of the nitrogenase reductase family protein for growth and pathogenicity of mycobacteria and answering this question will be one of our future aims.
In summary, by analysing 50 random mutants, we uncovered four genes from MAH to play a role in the interaction with host cells and thus in virulence. The homologues of three of the four genes were shown to contribute to virulence in other bacterial species, which supports the significance of our screening procedure.
Mutant complementation and evaluation of polar down-stream effects
To prove that the phenotypes of the mutants were indeed a cause of the inactivation of the mutated genes, we aimed at complementing the mutants by introducing the intact genes by electroporation. Only the transfer of gene MAV_3128 into the respective mutant was successful. Mutant MAV_3128 had shown the strongest and most different phenotypic changes in comparison to wild-type among the eight tested mutants in almost all the phenotypic tests. A complementation is best performed if the copy number of gene transcripts generated by the complementing gene narrows the copy number in the wild-type. We therefore used a plasmid for cloning (pMV306) that integrates once in the genome of the mutant and included the upstream region of MAV_3128 to most likely cover the promoter of the gene. This upstream region had a size of about 680 bp and the gene MAV_3127, which is located upstream of MAV_3128, has an orientation in opposite direction of MAV_3128 (see Figure 2). Therefore it was expected that the upstream region will contain the promoter sequence of the MAV_3128 gene. Thus a 3907 bp DNA fragment was cloned into the integrative vector pMV306. The resulting recombinant plasmid pFKaMAV3128 was successfully transformed into the mutant MAV_3128 to generate the complemented strain MAV3128Comp.
Selected phenotypic tests (plating on Congo Red Agar and intracellular survival) were repeated with the complemented strain. Upon plating on Congo Red agar (Figure 7 A), the pale colour of mutant MAV_3128 could no longer be seen in MAV3128Comp, except some pale corners in colonies. This may indicate the loss of the plasmid in absence of selection pressure. The intracellular survival experiment has also conclusively indicated a reversal of the mutation. The complemented strain showed more similar growth tendency towards wild-type strain than towards the mutant (Figure 7 B). In conclusion we successfully complemented the mutant MAV_3128 by introducing the intact gene proving that the phenotype of mutant MAV_3128 was indeed caused by the inactivation of gene MAV_3128 and not by a second line mutation.
Figure 7. Phenotype of the complemented strain MAV3128Comp compared to mutant MAV_3128 and WT. A: Colony morphology on Congo Red plates. B: Intracellular survival in human blood monocytes.
Since introduction of the intact genes into the other three mutants failed we additionally investigated the occurrence of polar effects in the four mutants by quantitative RT-PCR. As polar effects most probably will have an impact on genes which are located downstream of the mutated gene and exhibit the same orientation, we quantified expression of genes MAV_1779 (in mutant MAV_1778), MAV_3129 (in mutant MAV_3128), MAV_4332 (in mutant MAV_4334) and MAV_5105 (in mutant MAV_5106) by qRT-PCR. The 16S rRNA gene was used as reference gene. The ΔΔCT method was used to calculate expression of the gene in the corresponding mutant compared to the mean expression in the other three mutants. The expression levels measured were: MAV_1779 (in mutant MAV_1778): 2.1 fold, MAV_3129 (in mutant MAV_3128): 1.1 fold, MAV_4332 (in mutant MAV_4334): 1.0 fold and MAV_5105 (in mutant MAV_5106): 1.4 fold. In three of the four mutants, the expression of the down-stream genes transcribed in the same direction was not or only slightly changed. Only in mutant MAV_1778 a two-fold expression of gene MAV_1779 was observed. We conclude that with one exception no relevant polar effects could be observed.
Our study proposes a well-functioning method to randomly mutagenise MAH, by illegitimate recombination, genetically characterise the mutations to the nucleotide level and screen the mutants with simple phenotypic tests providing information about virulence-associated features.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
Conceived and designed the study: FAK and AL. Carried out the Laboratory work: FAK, AK, EK and RK. Manuscript drafted: FAK and AL. All authors read and approved the final manuscript.
We thank Dr. Elvira Richter, from National Reference Center for Mycobacteria, Borstel, Germany for generously providing 14 M. avium clinical isolates and Dr. Petra Möbius, from Friedrich Löffler Institute, Jena, Germany for giving 2 M. avium environmental strains. We also thank Prof. Dr. Michael Niederweis, University of Alabama, Birmingham, USA for donating plasmid pMN437.
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Microb Pathog 2009, 46:6-12. PubMed Abstract | Publisher Full Text
Stokes RW, Doxsee D: The receptor-mediated uptake, survival, replication, and drug sensitivity of Mycobacterium tuberculosis within the macrophage-like cell line THP-1: A comparison with human monocyte-derived macrophages.
Cell Immunol 1999, 197:1-9. PubMed Abstract | Publisher Full Text
Liu K, Yu J, Russell DG: pckA-deficient Mycobacterium bovis BCG shows attenuated virulence in mice and in macrophages.
Microbiology 2003, 149:1829-1835. PubMed Abstract | Publisher Full Text
Marrero J, Rhee KY, Schnappinger D, Pethe K, Ehrt S: Gluconeogenic carbon flow of tricarboxylic acid cycle intermediates is critical for Mycobacterium tuberculosis to establish and maintain infection.
Proc Natl Acad Sci U S A 2010, 107:9819-9824. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Meena LS, Chopra P, Bedwal RS, Singh Y: Cloning and characterization of GTP-binding proteins of Mycobacterium tuberculosis H37Rv.
Enzym Microb Technol 2008, 42:138-144. Publisher Full Text
Bijlsma JJE, Lie-A-Ling M, Nootenboom IC, Vandenbroucke-Grauls CMJE, Kusters JG: Identification of loci essential for the growth of Helicobacter pylori under acidic conditions.
J Infect Dis 2000, 182:1566-1569. PubMed Abstract | Publisher Full Text
Maloney E, Stankowska D, Zhang J, Fol M, Cheng QJ, Lun S, Bishai WR, Rajagopalan M, Chatterjee D, Madiraju MV: The two-domain LysX protein of Mycobacterium tuberculosis is required for production of lysinylated phosphatidylglycerol and resistance to cationic antimicrobial peptides.
PLoS Pathogens 2009, 5:e1000534. PubMed Abstract | Publisher Full Text | PubMed Central Full Text


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Alvin Liew, senior economist at UOB, explains what the slew of Japanese data released this week means for the Bank of Japan's upcoming policy meeting.» Read More
Most Asian currencies fell moderately on Friday, surrendering part of their gains from the previous session as investors fretted about the strength of inflation and prospects for slower economic growth.
The dollar rose Thursday, boosted by better-then-expected jobless claims data, but support remained fragile as record high oil prices stoked worries about the health of the U.S. economy.
The dollar fell to a one-month low against the euro Wednesday and touched a one-week trough versus the yen after the Federal Reserve lowered its growth estimates for 2008.
The dollar fell Tuesday as U.S. inflation data added to concerns about the economy's strength and raised doubt about whether the Federal Reserve will be able to hike interest rates this year.
The dollar rose on Monday, rebounding from a 2-1/2 week low against the euro as equities rallied and an economic forecasting gauge showed the U.S. economy, while weak, has so far managed to avoid recession.
For the week ending Friday, May 16, 2008, the U.S. Equity Markets ended the week up with all of the major indices up ~2% or more as stocks gained from M&A news, easing inflation worries, and strong earnings results. Oil and gasoline continued to hit new record highs as the dollar declined against major currencies.
The dollar extended losses against the euro and gave up gains versus the yen Friday, hurt by a report showing that U.S. consumer confidence tumbled to its lowest in 28 years in May.
The dollar fell in Europe Thursday as new data showed a drop in U.S. industrial production and unexpectedly strong first-quarter economic growth in Germany.
The dollar rebounded against the euro Wednesday as U.S. stocks extended gains and shrugged off a report showing tame reading on U.S. consumer prices in April.
The U.S. dollar rallied broadly Tuesday after a report on April retail sales beat forecasts and supported views that the Federal Reserve will probably stop cutting interest rates next month.
The dollar rose versus the Japanese yen and Swiss franc Monday as investors snapped up riskier assets such as stocks, encouraged by a dip in oil prices and unexpectedly strong earnings from HSBC.
For the week ending Friday, May 9, 2008, the U.S. Markets were negative for the week, with the Dow falling more than 200 points on Wednesday, making it the biggest point drop since 4/11/08.
The U.S. dollar pulled back from a two-month high against the euro after the European Central Bank left interest rates unchanged and its president's comments focused more on inflation than some had expected.
The dollar gained broadly on Wednesday, supported by hawkish comments from a Federal Reserve official that helped cement views that the U.S. central bank's cycle of aggressive interest rate cuts may be nearing an end.
The dollar extended declines against the euro and a basket of six major currencies for a second day Tuesday after earnings results from Fannie Mae came in weaker than expected.
The U.S. dollar slipped Monday against the euro but edged up against the British pound ahead of European Central Bank and Bank of England meetings later this week.
For the week ending Friday, May 9, 2008, the U.S. Markets were negative for the week, with the Dow falling more than 200 points on Wednesday, making it the biggest point drop since 4/11/08.
The dollar climbed to two-month peaks against the yen and a basket of currencies on Friday after a government report showed the U.S. economy shed just 20,000 jobs in April, fewer than economists had expected.
The dollar rose to fresh five-week highs against the euro Thursday after a survey showed a key U.S. manufacturing index for April came in slightly better than expected.
The currency trade has become increasingly popular as stock markets continue to move sideways and safe-haven government-backed bonds offer low yields.

subset: commoncrawl


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Hiroshima: A New Edition of John Hersey's Classic Story of Six People Who Lived Through The.....
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Hiroshima is the story of six human beings who lived through the greatest single manmade disaster in history. With what Bruce Bliven called "the simplicity of genius," John Hersey tells what these six — a clerk, a widowed seamstress, a physician, a Methodist minister, a young surgeon, and a German Catholic priest — were doing at 8:15 a.m. on August 6, 1945, when Hiroshima was destroyed by the first atomic bomb ever dropped on a city. Then he follows the course of their lives hour by hour, day by day.
The New Yorker of August 31, 1946, devoted all its space to this story. The immediate repercussions were vast: newspapers here and abroad reprinted it; during evening half-hours it was read over the network of the American Broadcasting Company; leading editorials were devoted to it in uncounted newspapers.
Almost four decades after the original publication of this celebrated book John Hersey went back to Hiroshima in search of the people whose stories he had told. His account of what he discovered about them — the variety of ways in which they responded to the past and went on with their lives — is now the eloquent and moving final chapter of Hiroshima.
"At exactly fifteen minutes past eight in the morning, on August 6, 1945, Japanese time, at the moment when the atomic bomb flashed above Hiroshima, Miss Toshiko Sasaki, a clerk in the personnel department of the East Asia Tin Works, had just sat down at her place in the plant office and was turning her head to speak to the girl at the next desk. At that same moment, Dr. Masakazu Fujii was settling down crosslegged to read the Osaka Asahi on the porch of his private hospital, overhanging one of the seven deltaic rivers which divide Hiroshima; Mrs. Hatsuyo Nakamura, a tailor's widow, stood by the window of her kitchen, watching a neighbor tearing down his house because it lay in the path of an air-raid-defense fire lane; Father Wilhelm Kleinsorge, a German priest of the Society of Jesus, reclined in his underwear on a cot on the top floor of his order's three-story mission house, reading a Jesuit magazine, Stimmen der Zeit; Dr. Terufumi Sasaki, a young member of the surgical staff of the city's large, modern Red Cross Hospital, walked along one of the hospital corridors with a blood specimen for a Wassermann test in his hand; and the Reverend Mr. Kiyoshi Tanimoto, pastor of the Hiroshima Methodist Church, paused at the door of a rich man's house in Koi, the city's western suburb, and prepared to unload a handcart full of things he had evacuated from town..."
A New edition of John Hersey's classic story of six people who lived through the explosion of the atomic bomb over Hiroshima in 1945--with an additional chapter in which the author, returning to Japan forty years later, tells us what has happened to each of these six survivors.
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OttLite Lighting High Definition 3-In-1 Craft Lamp
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$279.99 $223.99
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The versatile OttLite High Definition 3-in-1 Craft Lamp can be used as a floor lamp, clamp-on lamp or table lamp. Use the included hardware to convert this lamp into an illuminating element that's suitable for your work space. It features an adjustable magnifier lens and a gooseneck that can hold pictures, patterns, charts, instructions and more. This craft lamp is equipped with a wide shade for brilliant illumination and has a stylish design that complements any decor.
- Brand: Ottlite Lighting
- Dimensions: 45 inches high (min); 63.75 inches high (max); 12 x 8 inches diameter (base)
- Weight: 12.9 lbs


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Affordable housing is for people who are unable to rent or buy on the open market.
These pages explain what affordable housing is, what related products there are and how it is delivered.
Apply for affordable housing
To apply for affordable housing go to the HomeChoice website (housing register).
Affordable housing includes:
Homes to rent at:
- Affordable Rents.
- Social rents.
Homes to buy:
- Affordable home ownership.
Homes for older people:
- Sheltered housing for rent.
- Extra care housing for rent.
- Extra care affordable home ownership.
For developers and home builders looking to work with us to develop affordable housing options please see the relevant page in this section.
Affordable housing is for people who are unable to afford to rent or buy on the open market. There are different types of affordable housing schemes to suit people's needs.
To rent
- Affordable Rent – rent set at up to 80% of the local market rent, tenancies can be fixed term.
- Social rent – housing for rent based on a national rent formula, generally lower than Affordable Rent.
- Both the above are let through the local authority or a housing association.
To buy
Go to the Help to Buy South website or phone 0800 4561188. The schemes currently being offered are:
- Help to Buy Shared Ownership
- Help to Buy Equity Loan
- Help to Buy Rental schemes – Intermediate rent / Rent to Buy
For older households
- Sheltered housing – housing for older people, access is via HomeChoice Bristol website (housing register).
- Extra care housing for rent – sheltered housing with increased care and support.
- Extra care affordable home ownership – sheltered housing with increased care and support to buy. See our extra care housing page for both these extra care options.
- Retirement housing – see our Extra care housing page.
Who develops and provides affordable homes?
We work with our neighbouring councils, housing associations and private home builders to make affordable homes available. This partnership is known as the West of England Housing Delivery Panel.
Guide to the West of England Housing Delivery Panel (pdf, 117 KB)(opens new window).
Housing associations (also known as registered providers)
These are not for profit organisations that work with us to develop and manage affordable homes for rent and shared ownership.
Landlord Services
Our Landlord Services also have a development programme. These new homes plus any existing homes which become available are let through HomeChoice Bristol.
For details of local housing associations, Landlord Services and the properties they rent go to the HomeChoice Bristol website.
You can apply to bid for properties from all providers with just one application.
How do we help develop new affordable homes?
There are a number of ways, mainly:
- Working with the Homes and Communities Agency and Registered Providers to develop the Affordable Housing Programme
- We are also responsible for negotiating new affordable homes through the planning system on large new developments in the city
- Affordable Housing Framework – 2015-2020 (pdf, 0.6 MB)(opens new window)
- Housing Land Prospectus (pdf, 3.3 MB)(opens new window)
A new Affordable Housing Delivery Plan is being developed to increase the number of homes produced to up to 1,000 in the year 2016/17.
Short term loans for 'not for profit' organisations
Registered providers can now apply to Bristol City Council for an interest free, short term loan.
Specifically designed for 'not for profit' registered housing providers working in our city, interest free loans up to £50,000 per scheme are available for up to three years.
To find out more download the flyer and application form.
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Federal Power:
Additional Information on Repaying Federal Investments in Electric Power Facilities
RCED-86-44FS: Published: Nov 12, 1985. Publicly Released: Dec 12, 1985.
Additional Materials:
- Full Report:
(202) 512-4800
In response to a congressional request, GAO provided information on the basis of the Department of Energy's (DOE) practice of: (1) repaying the federal investment in power facilities within 50 years; and (2) charging an interest penalty when interest payments on the federal investment are deferred.
GAO found no documentation to explain the basis for the determination that 50 years is a reasonable period in which to amortize federal investment in a project; however, this period has become a well-established administrative practice and, in authorizing certain individual projects, Congress has specified a repayment period of 50 years. In addition, GAO found that DOE and Department of the Interior orders require interest to be charged on deferred interest, and Congress has viewed the interest penalty as an incentive for timely repayment.
Oct 1, 2015
Spent Nuclear Fuel:Legislative, Technical, and Societal Challenges to Its TransportationGAO-16-121T: Published: Oct 1, 2015. Publicly Released: Oct 1, 2015.
Sep 23, 2015
Nuclear Nonproliferation:DOE Made Progress to Secure Vulnerable Nuclear Materials Worldwide, but Opportunities Exist to Improve Its EffortsGAO-15-799: Published: Sep 23, 2015. Publicly Released: Sep 23, 2015.
Jul 28, 2015
Technology Assessment:Nuclear Reactors: Status and Challenges in Development and Deployment of New Commercial ConceptsGAO-15-652: Published: Jul 28, 2015. Publicly Released: Jul 28, 2015.
Jul 27, 2015
Defense Energy:Observations on DOD's Investments in Alternative FuelsGAO-15-674: Published: Jul 27, 2015. Publicly Released: Jul 27, 2015.
Jul 13, 2015
DOE Project Management:NNSA Should Ensure Equal Consideration of Alternatives for Lithium ProductionGAO-15-525: Published: Jul 13, 2015. Publicly Released: Jul 13, 2015.
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Check-Off Programs:Observations from the Propane and Heating Oil ProgramsGAO-15-769T: Published: Jul 10, 2015. Publicly Released: Jul 10, 2015.
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Crude Oil Export Restrictions:Studies Suggest Allowing Exports Could Reduce Consumer Fuel PricesGAO-15-745T: Published: Jul 8, 2015. Publicly Released: Jul 8, 2015.
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Electricity:Generation Mix Has Shifted, and Growth in Consumption Has Slowed, Affecting System Operations and PricesGAO-15-524: Published: May 29, 2015. Publicly Released: Jun 29, 2015.
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Renewable Energy:BLM Has Limited Assurance That Wind and Solar Projects Are Adequately BondedGAO-15-520: Published: Jun 5, 2015. Publicly Released: Jun 23, 2015.
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Energy Savings Performance Contracts:Additional Actions Needed to Improve Federal OversightGAO-15-432: Published: Jun 17, 2015. Publicly Released: Jun 17, 2015.
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The North Ridgeville Baseball League, Inc. recognizes an outstanding volunteer each year by bestowing "The Jacket Award." It takes an army of volunteers each season to make the league a success and the leagues wishes to honor those who go above and beyond in making that a reality.
Nominations will be accepted until September 2, 2015. You may nominate as many deserving people as you like. All nominations will be reviewed by an independent committee and the winner will be named at the September coaches' meeting.
The winner will receive a customized jacket as well as recognition on a plaque and through electronic media. All nominees will also be recognized. Please take a moment to nominate that special person or persons that really went all out this year!
Please click on the link below to submit your nomination(s).
The Registration "2015 Jacket Award" is not currently available.
This Registration closed on 09/03/2015, 11:45pm EDT (EDT).
We will be hosting fall clinics for those boys moving up to play Class HH (9U) or Class H (10U) in 2016! These sessions are free to all players from the 2015 season and will be held on Fridays, starting 8/28 through 10/2 (no clinic Labor Day Weekend.) Please see the flyer below. No registration is required, so please just show up to as many clinics as you desire! For questions, contact Chris Iezzi (Yet'-zee) at 216.392.5252.
We are also proud to announce that we will be hosting girls softball clinics every Wednesday (weather permitting) from 6:00 until 7:00 PM at Palmer Field for all girls, ages 5-18! The cost is free! Different skill sets will be worked each week and you can attend any clinic you wish. For questions, contact Joel Melvin at 440.610.1970. We look forward to seeing you there!
2016 Head Coaching Applications
Want to be a head coach for the 2016 season? Click below to register to become a head coach. All applications will be reviewed by the Board. Interviews will then be scheduled at a mutually convenient time.
Complete the online registration today!
Download the free Sport Ngin app for Android and Apple devices, or follow us on the web with your PC or mobile browser! Once you create an account using only your email address and unique password, you'll be able to follow your team or league schedule, standings, or scores throughout the season. You can use the team ID that appears in the top right corner of each team's page to find them on the app or you can search for them by name. Coaches will be able to make updates to the scores with the touch of their screens!
You can make the selections through the tag filter for the teams or leagues that you want to see on the calendar or you can see all of the games going on throughout the North Ridgeville Baseball League. You can also copy the URL of the feed (by clicking the "Subscribe to iCal feed" button below the calendar), with or without your tag filters, into your Outlook or Google calendars - or you can follow the instructions from our document page to be able to add the calendar feed to your Apple device. Instant information and change notifications are just a few clicks away!
The girls clinic will be canceled for this evening (9/30). We will resume next Wednesday (10/7) from 6-7PM.
This is good... http://t.co/HvzPRbUZPa
This week's (9/23) girls softball skills clinic is canceled. We will resume the clinics next Wednesday 9/30 from 6-7PM.


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You gotta get to this jardy its so sick!
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Baxter Street renewal: New tenants, changes to commercial corridor help to revive interest in the Baxter Street business district
The new Athens Neurological Associates building has contributed to the revitalization of Baxter Street.
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We now have new chrome lined US replacement barrels available. They have the
extractor cut and a hot salt black oxide finish. They come with new pins and the
necessary drills for installing them. They are $169.00
We also are offering barrel building services for $75.00, we will install your parts ,
Pin, drill gas port and hot salt finish them, for $75.00.


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I am very talented and creative individual, completely dedicated to my job, that I truly LOVE! I work alone, I do not have a group of people who works for me, as this is the only way I can guarantee for the quality of my work. I am an all around Media expert and I have 8 years of experience in video editing. Provide quality services to clients. Adobe Premiere Pro, Adobe After Effect , Videohive AE templates Modification, Slideshow, Powerpoint Videos, Camtasia Editing, Whiteboard Animation Video, Adobe PhotoShop, Microsoft PowerPoint and more.. I have been working as a video editor in post-production for the past 8 years. I learn quickly, and I am able to adapt my skills to fit a variety of needs and projects. If you require any professionally edited video, I will go above and beyond your needs and expectations. I can take your projects from conception to completion with the utmost speed and accuracy with superb creativity and quality. Clients satisfaction is my utmost priority. I like to tackle projects individually giving each project and each of my clients my undivided attention. I look forward to work with you.
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According to a recent filing by the whistleblowers: "The government has not joined in Merck's current motion [to dismiss the case] and has made no decision on the current Complaint. Instead, it has taken a 'wait and see' approach requesting that it be served with all pleadings, motions and court orders in this case, and that its consent be obtained before the case is settled, dismissed or discontinued.
"While the Department of Justice has chosen to sit on the sidelines of this case for now, both the FDA and its sister agency, the National Institute of Health, have since the complaint was filed begun to take steps to address the failure of Merck's mumps vaccine. The FDA has initiated its own study to determine the vaccine's efficacy, acknowledging that the recent mumps outbreak 'indicat[es] lower vaccine efficacy than previously estimated.' The NIH has gone even further. It is funding the University of Georgia to develop a new mumps vaccine because the recent outbreaks 'strongly suggest that the current vaccine is not effective.'"
Merck continues to state that its mumps vaccine is highly effective, as shown in this ad filed with the court by the whistleblowers; it claims 96 percent efficacy for the mumps portion of the MMRII -- in other words, 96 out of 100 people who get the shot are protected from the mumps virus:
Merck could face billions in punitive damages or even lose its license to manufacture the vaccine if it were convicted of defrauding the government, which has spent hundreds of millions of dollars to buy the MMR II for mass vaccination campaigns. At the very least, a new MMR vaccine based on a different mumps component would cut into the market share of Merck's MMRII. The shot is a mainstay of the recommended childhood vaccination schedule -- and of company profits.
Also at risk is the health of anyone who comes down with mumps at a later age than the disease usually occurs in childhood. When contracted early it is usually benign, but caught later it can be far more serious. In males it can be excruciatingly painful and even lead to sterility.
http://www.cdc.gov/mmwr/preview/mmwrhtml/mm6217md.htm?s_cid=mm6217md_wOverall, the numbers are up by more than 60 percent so far in 2013, a figure consistent with a spike but not a major epidemic. The situation in the South Atlantic region shows a bigger discontinuity, with more than 80 cases reported this year, tenfold the number for all of 2012. WTOP in Washington reported May 3: "The college years seem to be a prime time for coming down with the mumps. This spring, outbreaks of mumps have been reported at three universities in Virginia: the University of Richmond, Virginia Commonwealth University and James Madison University. Cases have also been reported at Loyola University in Baltimore.
"'The outbreaks at area college campuses are part of a national pattern,' says Dr. David Hyun, an infectious disease specialist at Children's National Medical Center. 'The outbreaks that we have seen in the past few years tend to come from college students,' he says, adding the prime age for mumps now seems to be 15 to 24.
"No one is exactly sure why. But Hyun says the mumps vaccine could be losing its effectiveness over time."
That, of course, is consistent with the claims in the whistleblower suit, which was unsealed in U.S. District Court in Philadelphia last June. As we reported then, the alleged fraud was a multi-year effort to hide the fact that the mumps vaccine is no longer anywhere near as effective as Merck claims. Project 007 was widely known and approved within the company's vaccine division, according to the two former Merck scientists who filed the suit under the federal whistleblower statute.
Virologists Stephen A. Krahling and Joan A. Wlochowski claim they witnessed the fraud firsthand when they worked at the Merck vaccine laboratory in West Point, Pennsylvania, between 1999 and 2002, and were pressured to participate.
They describe a supervisor frantically changing test results that showed the vaccine wasn't working; destroying garbage-bags full of evidence to keep the fraud from being exposed; and lying to FDA regulators who came to the lab after being alerted by the whistleblowers.
A top Merck vaccine official told Krahling the matter was a "business decision," the suit says, and he was twice told the company would make sure he went to jail if he told federal regulators the truth.
The alleged fraud occurred because, in order to maintain its license for the mumps-measles-rubella vaccine, known as the MMRII, Merck needed to show that the mumps vaccine was still as potent as when originally approved in 1967 as a single vaccine, able to induce immunity in 95 percent of those vaccinated. That number, according to vaccine authorities, is crucial because it leads to "herd immunity," protective against outbreaks even among unvaccinated people.
The problem with the mumps vaccine lay in the fact that by the late 1990s, after decades of producing it with the original strain of mumps virus, the vaccine's effectiveness had steadily declined, the suit says.
Merck is the only company licensed in the United States to produce the individual mumps vaccine, as well as the MMRII and a newer shot called the MMRV or ProQuad, which also contains the chickenpox vaccine. That gives Merck an effective monopoly on the product line, which by our estimate has brought the company more than $10 billion in business since 2000. The complaint conservatively estimates MMRII purchases by the Centers for Disease Control and Prevention at $750 million.
We noted that weak efficacy could be triggering real-time, real-world health problems here and abroad, where a version of the MMRII is also used. Mumps outbreaks unexpectedly occurred in the United States in 2006 and in 2009-10, reflecting the three-year cycle in which younger children become exposed. A total of 6,500 cases were reported in a highly vaccinated population in the Midwest in 2006, according to the suit, and another 5,000 cases in 2009; in the years leading up to the first outbreak, the annual average had been 265 cases.
That three-year cycle could be repeating itself, judging from the recent reports in Virginia, Maryland, and Massachusetts. Despite the fact the CDC surveillance data is not showing large numbers, one possibility is the surveillance is actually missing a surge in mumps cases, many of which are not reported to health authorities. The CDC acknowledges the difficulty of confirming mumps outbreaks in heavily vaccinated populations, stating that "persons with a history of mumps vaccination may not have detectable mumps ... antibody." (An Age of Autism reader who was vaccinated during childhood with MMRII, two weeks after spending time in one of the cluster areas reported to us the characteristic symptoms of mumps. By then, thousands of miles away from the cluster, this reader simply chose to stay at home sick and did not report the condition, seek testing or medical treatment.)
The whistleblower case is now subject to the usual blizzard of motions in federal court, with Merck demanding that the case be dismissed. The Justice Department has so far not joined the lawsuit on the whistleblower's behalf, but that remains a possibility. Merck has filed a motion to have the case dismissed; the whistleblowers, called "relators" in the suit, responded with a document explaining what they believe is at stake:
"This case involves Merck's efforts to conceal from the government and the public that Merck's forty-five year old mumps vaccine no longer provides adequate immunization. Relators know this because they were virologists in the Merck lab at the center of this fraudulent campaign. They were even pressured by their superiors to participate in the scheme, which included, among other things, falsifying test data; destroying evidence; lying to the FDA; and most importantly, continuously hiding from the government and the public over the course of a decade -- including through two unprecedented mumps outbreaks -- the significantly diminished efficacy of the vaccine.
"Relators know this not only because of their eyewitness account of the activity. They also know it because the Merck executives that led the campaign admitted it to them. They admitted that Merck's vaccine had significantly degraded through Merck's repeated passaging of it for what is now approaching five decades, to make more and more vaccine for the millions of children that take it every year. They admitted that this degradation would eventually lead to the reemergence of mumps outbreaks, which it has. Worst of all, they admitted that Merck engaged in this fraud and deception as a 'business decision' necessary to ensure that Merck maintained its exclusive license to sell the vaccine in the U.S. "
"Mumps may seem like a disease of a bygone era to many people in the U.S. who, thanks to immunization programs, have been spared the fever, aches and characteristic swollen jawline of the once common viral infection," according to the university news agency.
"Biao He, a University professor of infectious diseases and a Georgia Research Alliance distinguished investigator in the College of Veterinary Medicine, worries that a new strain of the virus is spreading, and it could lead to the widespread reintroduction of mumps.
"Now, thanks in part to a $1.8 million grant from the National Institutes of Health, He and his team are working on a new vaccine to stop it."
Dan Olmsted is Editor and Mark Blaxill is Editor at Large of AgeOfAutism.com. They are co-authors of The Age of Autism – Mercury, Medicine, and a Man-made Epidemic, published in 2010 by Thomas Dunne Books.
Posted by Age of Autism at May 14, 2013 at 5:45 AM
in Dan Olmsted , Dan Olmsted , Mark Blaxill
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If Cruz wins the November election, Senator Cornyn has this advice — remember to work well with others.
"The Senate, as any authoritarian body would be, depends on people's ability to persuade and to achieve consensus and to get a majority vote in order to make policy. So it's a little different job, and I know Ted understands that and appreciates that."
Cruz faces former Texas state representative Paul Sadler, the winner of the Democratic runoff, in the November election.