Reverse transcriptase-PCR differential display analysis of meningococcal transcripts during infection of human cells: Up-regulation of and its role in intracellular replication-8

R infection. Antibodies against were used before permeabilization with saponin in combination with a secondary antibody conjugated with Cy5 and, after permeabilization, with a secondary TRITC-conjugated antibody. Intracellular bacteria were therefore red while extracellular bacteria were expected to be purple (the combination of TRITC and Cy5 signal). In the images all bacteria were intracellular. To detect cellular markers we used anti-LAMP1 and a secondary antibody conjugated with FITC.<p><b>Copyright information:</b></p><p>Taken from "Reverse transcriptase-PCR differential display analysis of meningococcal transcripts during infection of human cells: Up-regulation of and its role in intracellular replication"</p><p>http://www.biomedcentral.com/1471-2180/8/131</p><p>BMC Microbiology 2008;8():131-131.</p><p>Published online 29 Jul 2008</p><p>PMCID:PMC2527323.</p><p></p

Reverse transcriptase-PCR differential display analysis of meningococcal transcripts during infection of human cells: Up-regulation of and its role in intracellular replication-5

the Live/Dead Bacbacterial viability kit (Molecular Probe). (A) Representative images of Live/Dead-stained meningococci: live bacteria fluoresce green, while dead bacteria fluoresce red. (B) Percentages of non-viable meningococci were equal to ratios between bacteria staining positive with propidium iodide (red) and the total stained (red + green) bacteria. Data are shown as mean ± standard deviation of three experiments. Statistically significant difference between values from B1940 (or B1940ΩpriA/priA+) and B1940ΩpriA (asterisk) is declared at a p value < 0.05.<p><b>Copyright information:</b></p><p>Taken from "Reverse transcriptase-PCR differential display analysis of meningococcal transcripts during infection of human cells: Up-regulation of and its role in intracellular replication"</p><p>http://www.biomedcentral.com/1471-2180/8/131</p><p>BMC Microbiology 2008;8():131-131.</p><p>Published online 29 Jul 2008</p><p>PMCID:PMC2527323.</p><p></p

Reverse transcriptase-PCR differential display analysis of meningococcal transcripts during infection of human cells: Up-regulation of and its role in intracellular replication-2

Ver and the probe (open rectangle) used in the Southern blot experiments shown in panels B and C. , erythromycin-resistance gene; DUS, DNA uptake sequence; B, HI restriction sites; Hf, fI restriction sites. (B) Southern blot analysis demonstrating the inactivation of . Chromosomal DNAs were extracted from the parental strain B1940 (lane 4) and from three recombinant strains (B1940ΩpriA) transformed with pDEXpriA (lanes 1–3). The fI-restricted chromosomal DNAs were analyzed by Southern blot using the P labeled -specific DNA fragments cloned in pDEXpriA as probes. The arrows on the right indicate -specific fragments whose sizes were deduced on the basis of the relative migration of DNA ladders (bars on the left). (C) Southern blot experiment demonstrating complementation of B1940ΩpriA. B1940ΩpriA was transformed with pACpriA harboring a functional copy of , and chromosomal DNAs were extracted from two recombinant strains (B1940ΩpriA/priA+) (lanes 3 and 4). The DNAs from these two strains, from B1940 (lane 1) and from B1940ΩpriA (lane 2) were digested with fI and analyzed as in panel B.<p><b>Copyright information:</b></p><p>Taken from "Reverse transcriptase-PCR differential display analysis of meningococcal transcripts during infection of human cells: Up-regulation of and its role in intracellular replication"</p><p>http://www.biomedcentral.com/1471-2180/8/131</p><p>BMC Microbiology 2008;8():131-131.</p><p>Published online 29 Jul 2008</p><p>PMCID:PMC2527323.</p><p></p

Reverse transcriptase-PCR differential display analysis of meningococcal transcripts during infection of human cells: Up-regulation of and its role in intracellular replication-4

He typical diplococcal, coffee bean-shaped morphology of meningococci.<p><b>Copyright information:</b></p><p>Taken from "Reverse transcriptase-PCR differential display analysis of meningococcal transcripts during infection of human cells: Up-regulation of and its role in intracellular replication"</p><p>http://www.biomedcentral.com/1471-2180/8/131</p><p>BMC Microbiology 2008;8():131-131.</p><p>Published online 29 Jul 2008</p><p>PMCID:PMC2527323.</p><p></p

Reverse transcriptase-PCR differential display analysis of meningococcal transcripts during infection of human cells: Up-regulation of and its role in intracellular replication-1

Rol bacteria grown for 7 h in culture medium (control) and were used to generate gene-specific P-labeled cDNA probes as detailed in the Methods section. The P-labeled cDNA probes were hybridized to different amounts (4, 20 and 100 ng) of denatured NMB0551 ()-specific DNA fragments. For the 16S rRNA gene-specific fragment, two-fold serial dilutions (from 0.05 to 50 ng) were used. (B) Densitometry analysis of the RT-PCR slot blot with 20 ng of -specific DNA fragments. The relative transcript levels of in intracellular meningococci are arbitrarily assumed equal to 100%. Values represent means from five independent experiments, each with triplicate samples, using RNA preparations from distinct infection assays. Bars indicate standard deviations. (C) Semi-quantitative analysis of the -specific transcripts by RT real-time PCR experiment. The RNAs were extracted from intracellular or control meningococci as described above. Results were normalized to 16S rRNA levels. Transcript levels of in intracellular meningococci were arbitrarily given a value of 100%. Data are shown as mean ± standard deviation from five independent experiments, each with triplicate samples, using RNA preparations from distinct infection assays. The Student's T-test was used for statistical analysis. Statistically significant differences between values from intracellular and control bacteria (asterisks) are declared at a p value < 0.05.<p><b>Copyright information:</b></p><p>Taken from "Reverse transcriptase-PCR differential display analysis of meningococcal transcripts during infection of human cells: Up-regulation of and its role in intracellular replication"</p><p>http://www.biomedcentral.com/1471-2180/8/131</p><p>BMC Microbiology 2008;8():131-131.</p><p>Published online 29 Jul 2008</p><p>PMCID:PMC2527323.</p><p></p

Reverse transcriptase-PCR differential display analysis of meningococcal transcripts during infection of human cells: Up-regulation of and its role in intracellular replication-6

Ed times to hydrogen peroxide (A) or nitric oxide generator sodium nitroprusside (B) in GC liquid medium with shaking and then viability was determined by recovery of viable bacteria. Data are shown as mean ± standard deviation from three independent experiments, each with triplicate samples. Statistically significant differences between values from B1940 and B1940ΩpriA (asterisks) are declared at a p value < 0.05.<p><b>Copyright information:</b></p><p>Taken from "Reverse transcriptase-PCR differential display analysis of meningococcal transcripts during infection of human cells: Up-regulation of and its role in intracellular replication"</p><p>http://www.biomedcentral.com/1471-2180/8/131</p><p>BMC Microbiology 2008;8():131-131.</p><p>Published online 29 Jul 2008</p><p>PMCID:PMC2527323.</p><p></p

Reverse transcriptase-PCR differential display analysis of meningococcal transcripts during infection of human cells: Up-regulation of and its role in intracellular replication-0

Nthesis using total RNAs from intracellular meningococci (strain B1940) recovered from saponin-lysed HeLa cells after 7 h of infection (intracellular) or control bacteria grown for 7 h in cell culture medium without HeLa cells (control) as templates. Then second-strand cDNAs synthesis was carried out using the corresponding oligonucleotides and a mixture of random hexamers as primers, and the PCR products were analyzed by polyacrylamide gel electrophoresis. In lanes 1 and 6 molecular weight ladders, whose sizes are indicated on the left of each panel, were run in parallel. The arrows indicate bands corresponding to either up-regulated (, , , , , , , , , ) or down-regulated genes () in the intracellular environment. Molecular weight DNA ladders were run in parallel. (B) Limited transcriptional analysis. A partial AI-restricted genomic library from the strain B1940 was constructed. Individual plasmid clones were digested with AI and a Southern blot analysis was performed using P-labeled cDNA probes derived from intracellular bacteria after 8 h of infection of HeLa cells or control bacteria grown for 8 h in cell culture medium. In the figure only a limited number of clones (22 clones out of 3000) on duplicate filters are shown. The arrow indicates the cloned NMB0551 ORF corresponding to , up-regulated by intracellular meningococci. Asterisks mark the 983 and 1123 bp-long 3AI fragments contained in the plasmid clone. (C) Genetic map of meningococcal genes up-regulated in the intracellular environment. The positions of the cDNAs (closed rectangles) corresponding to up-regulated genes that were identified by RT-PCR-DD () are indicated with respect to the available genetic map of MC58. Open rectangles with asterisks locate the 983 and 1123 bp-long 3AI fragments contained in the plasmid clone shown in panel B.<p><b>Copyright information:</b></p><p>Taken from "Reverse transcriptase-PCR differential display analysis of meningococcal transcripts during infection of human cells: Up-regulation of and its role in intracellular replication"</p><p>http://www.biomedcentral.com/1471-2180/8/131</p><p>BMC Microbiology 2008;8():131-131.</p><p>Published online 29 Jul 2008</p><p>PMCID:PMC2527323.</p><p></p

Reverse transcriptase-PCR differential display analysis of meningococcal transcripts during infection of human cells: Up-regulation of and its role in intracellular replication-9

Nthesis using total RNAs from intracellular meningococci (strain B1940) recovered from saponin-lysed HeLa cells after 7 h of infection (intracellular) or control bacteria grown for 7 h in cell culture medium without HeLa cells (control) as templates. Then second-strand cDNAs synthesis was carried out using the corresponding oligonucleotides and a mixture of random hexamers as primers, and the PCR products were analyzed by polyacrylamide gel electrophoresis. In lanes 1 and 6 molecular weight ladders, whose sizes are indicated on the left of each panel, were run in parallel. The arrows indicate bands corresponding to either up-regulated (, , , , , , , , , ) or down-regulated genes () in the intracellular environment. Molecular weight DNA ladders were run in parallel. (B) Limited transcriptional analysis. A partial AI-restricted genomic library from the strain B1940 was constructed. Individual plasmid clones were digested with AI and a Southern blot analysis was performed using P-labeled cDNA probes derived from intracellular bacteria after 8 h of infection of HeLa cells or control bacteria grown for 8 h in cell culture medium. In the figure only a limited number of clones (22 clones out of 3000) on duplicate filters are shown. The arrow indicates the cloned NMB0551 ORF corresponding to , up-regulated by intracellular meningococci. Asterisks mark the 983 and 1123 bp-long 3AI fragments contained in the plasmid clone. (C) Genetic map of meningococcal genes up-regulated in the intracellular environment. The positions of the cDNAs (closed rectangles) corresponding to up-regulated genes that were identified by RT-PCR-DD () are indicated with respect to the available genetic map of MC58. Open rectangles with asterisks locate the 983 and 1123 bp-long 3AI fragments contained in the plasmid clone shown in panel B.<p><b>Copyright information:</b></p><p>Taken from "Reverse transcriptase-PCR differential display analysis of meningococcal transcripts during infection of human cells: Up-regulation of and its role in intracellular replication"</p><p>http://www.biomedcentral.com/1471-2180/8/131</p><p>BMC Microbiology 2008;8():131-131.</p><p>Published online 29 Jul 2008</p><p>PMCID:PMC2527323.</p><p></p

Reverse transcriptase-PCR differential display analysis of meningococcal transcripts during infection of human cells: Up-regulation of and its role in intracellular replication-7

A were centrifuged onto cells to start infection. After saponin lysis, CFU from intracellular bacteria were scored. Values are relative to the number of intracellular B1940 at time 0 (10bacteria per 2 × 10cells per ml) and are means of at least ten independent experiments made in triplicate with standard errors.<p><b>Copyright information:</b></p><p>Taken from "Reverse transcriptase-PCR differential display analysis of meningococcal transcripts during infection of human cells: Up-regulation of and its role in intracellular replication"</p><p>http://www.biomedcentral.com/1471-2180/8/131</p><p>BMC Microbiology 2008;8():131-131.</p><p>Published online 29 Jul 2008</p><p>PMCID:PMC2527323.</p><p></p

Calcite-forming <i>Bacillus licheniformis</i> Thriving on Underwater Speleothems of a Hydrothermal Cave

<p>The underwater environment of Grotta Giusti (Monsummano Terme, Italy) is a suggestive setting with different types of speleothems including “leafy” and “cauliflower” concretions along the walls and roof, and conical pseudo-stalagmites on the floor. Very high calcium and dissolved CO₂ levels, and massive calcium carbonate precipitation characterize this cave environment. Yet, life thrives on the leafy concretion surfaces with loads of cultivable heterotrophic microorganisms around 10⁵ colony-forming units per cm². <i>Bacillus licheniformis</i> appeared to be the prevalent cultivable microorganism on a low-nutrient medium that was used for screening. 16S rRNA gene-based polymerase chain reaction–single strand conformation polymorphism profiling indicated that Group VI <i>Bacillaceae</i> species was well represented in the bacterial community of underwater speleothems. Interpretation of X-ray diffraction spectra and Raman spectroscopy data indicated that the <i>B. licheniformis</i> isolate produced <i>in vitro</i> abundant calcite microcrystals that were also characterized by scanning electron microscopy coupled with energy dispersive X-ray spectroscopy. Production of calcite microcrystals was analyzed in different media (Christensen’s urea agar and B4 calcium carbonate precipitation medium) and incubation conditions, and it was found to be enhanced by nitrate supplement in B4 medium under low-oxygen conditions. B4 and B4-nitrate media also stimulated antibiotic production by the <i>B. licheniformis</i> isolate, which was analyzed by microbiological assays.</p