Virulence of Bacillus cereus as natural facultative pathogen of Anopheles subpictus Grassi (Diptera: Culicidae) larvae in submerged rice-fields and shallow ponds

Out of 4407 Anopheles subpictus larvae collected from submerged rice-fields and shallow ponds, 1412 were found to be unhealthy and 2.8% of unhealthy larvae were naturally infected by Bacillus cereus. B. cereus formed circular, white and flat colonies. Bacteria were gram positive, ellipsoidal/oval spore forming aerobic rods. Although the isolate was positive for catalase, urease, gelatinase, lipase, nitrate reduction and H2S production, it was negative for indole production, Vogues-Proskauer test, oxidase test and acid/gas production from carbon sources. Through biochemical characterization and fatty acid methyl ester (FAME) analysis, the bacterial isolate was identified as Bacillus cereus. In the laboratory condition, B. cereus suspension resulted in 43.57% and 93.78% death of A. subpictus larvae within 3 and 6 h, respectively. The organisms were sensitive to recommended doses of kanamycin, gatifloxacin, gentamycin, levofloxacin, doxycyclin,  tetracyclin, streptomycin, rifampicin, vancomycin, ciprofloxacin, but found resistant to ampicillin.Key words: Bacillus cereus, fatty acid methyl ester analysis, scanning electron micrograph, biochemicalcharacterization, pathogen, Anopheles subpictus larva

Detection of S-nitrosothiol and nitrosylated proteins in Arachis hypogaea functional nodule: response of the nitrogen fixing symbiont.

To detect the presence of NO, ROS and RNS in nodules of crack entry legumes, we used Arachis hypogaea functional nodule. The response of two cognate partner rhizobia was compared towards NO and GSNO using S. meliloti and Bradyrhizobium sp NC921001. ROS, NO, nitrosothiol and bacteroids were detected by fluorescence microscopy. Redox enzymes and thiol pools were detected biochemically. Nitrosothiols were found to be present but ROS and NO were absent in A. hypogaea nodule. A number of S-nitrosylated proteins were also detected. The total thiol pool and most of the redox enzymes were low in nodule cytosolic extract but these were found to be high in the partner microorganisms indicating partner rhizobia could protect the nodule environment against the nitrosothiols. Both S. meliloti and Bradyrhizobium sp NC921001 were found to contain GSNO reductase. Interestingly, there was a marked difference in growth pattern between S. meliloti and Bradyrhizobium sp in presence of sodium nitroprusside (SNP) and S-nitrosoglutathione (GSNO). Bradyrhizobium sp was found to be much more tolerant to NO donor compounds than the S. meliloti. In contrast, S. meliloti showed resistance to GSNO but was sensitive to SNP. Together our data indicate that nodule environment of crack entry legumes is different than the nodules of infection mode entry in terms of NO, ROS and RNS. Based on our biochemical characterization, we propose that exchange of redox molecules and reactive chemical species is possible between the bacteroid and nodule compartment

Biochemical quantification of Nitrosothiol by Saville assay.

<p>Quantification of nitrosothiol in 22, 40, 50, 65 and 78 day old nodule extracts of <i>A. hypogaea</i> was done by Saville assay. Results were expressed as mean ± SD, for n = 3 experiments. P≤0.01, using one-way ANOVA.</p

Quantification of thiol pool in <i>S. meliloti</i> 1021 and <i>Bradyrhizobium sp</i> after 30 minute stress with SNP in media.

<p><i>S. meliloti</i> cells were grown to mid log phase at 30°C in TY media whereas <i>Bradyrhizobium sp</i> cells were grown similarly in YEM media. Sodium nitroprusside (SNP) was added to mid log phase cell in the final concentration of 1.0 and 3.0 mM and incubated at 30°C for 30 minute. Cells were harvested, lysed and cell free crude extracts were subjected to quantification of total thiol, oxidized glutathione, reduced glutathione and ratio for reduced to oxidized glutathione. Results are expressed as mean ± SD, for n = 3 experiments. P≤0.01, using one-way ANOVA.</p

NO detection in <i>A. hypogaea</i> JL 24 and <i>M. sativa</i> nodules.

<p>All nodule sections were incubated with 10 µM DAF-2DA at 25°C for 1 hour in darkness. Photographs are representative of results obtained from the analysis of nodules in five independent experiments. Images of <i>A. hypogaea</i> nodule sections show absence of NO dependent DAF-2DA fluorescence (green colour) in (A) 20 day, (C) 40 day and (E) 80 day old nodules. (B), (D) and (F) show the corresponding bright field images. Scale bar = 200 µm. Whereas DAF-2DA dependent green colour fluorescence was observed in (G) 143 day old <i>M. sativa</i> nodule section. (H) represents the corresponding bright field image. Scale bar = 100 µm. The images of the sections were captured using excitation at 485 nm and emission at 530 nm.</p

Representation of maintenance of Redox Cycle in nodules and bacteroid compartments.

<p>Scheme represents the nodule redox environment, and transport of redox molecules in plant cell compartment and bacteroid compartment. Based on the biochemical analysis of thiols, RSNO and redox active enzymes of plant cell and rhizobia, the exchange of molecules between the compartments has been shown. Upward arrow head indicates upregulation, downward arrowhead indicates downregulation. RSNO could be produced in the nodule compartment by several routes. Reduced glutathiones (GSH) take part in transnitrosylation reaction in nodule compartment. GSNO produced in nodule compartment may diffuse to the bacteroid cell where GSNO reductase could convert it to GSSG and NH₃; GSSG is recycled by GR and GPx. High basal level GR present in the bacteroid could recycle GSSG to GSH. GSH from the bacteroid may be transported to the nodule compartment to take part in the trans nitrosylation reaction. H₂O₂ produced in nodule compartment could be detoxified by nodule catalase, and APx and the excess H₂O₂ might be diffused to the bacteroid compartment where <i>Bradyrhizobium</i> could detoxify it with high level of catalase and GPx.</p

Effect of sodium nitroprusside (SNP) and GSNO on the growth of <i>S. meliloti</i> and <i>Bradyrhizobium sp</i>.

<p>Growth curves of <i>S. meliloti</i> (A) and <i>Bradyrhizobium sp</i> (B) in the presence of different concentrations of SNP: Mid-log phase cells (OD₅₉₀ = 0.5–0.7) were diluted in fresh medium containing graded concentration of SNP (—◊— control, —□— 0.5 mM, —Δ— 1.0 mM, and —×— 3.0 mM) and were grown under their respective growth conditions and media. Same procedures were performed to study the effect of GSNO (—◊— control, —□— 0.5 mM, —Δ— 1.0 mM, and —×— 3.0 mM) on growth of <i>S. meliloti</i> (C) and <i>Bradyrhizobium sp</i> (D).</p

Detection of S-nitrosylated proteins in <i>A. hypogaea</i> nodules.

<p>Bacteroid free nodule extracts of 20, 40 and 80 day were processed as per described in the S-nitrosylated protein detection assay kit of Cayman Chemicals, Ann Arbor, Michigan with slight modification. Gel profile of the blot is representative of results obtained from the analysis of three independent experiments. (A) Lane 1, 2 and 3 depict reduced and biotinylated samples of 20, 40 and 80 day old nodule extracts. Lane 4 represents the marker. Lane 5, 6 and 7 represent the endogenously biotinylated protein profiles correspond to lanes 1, 2 and 3. (B) is the corresponding gel profile of blot (A).</p

RSNO fluorescence intensity profile of nodules from <i>A. hypogaea</i> JL 24 and <i>M. sativa</i>.

<p>The mean intensity of green colour (as observed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045526#pone-0045526-g002" target="_blank">Figure 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045526#pone-0045526-g003" target="_blank">3</a>) of nodule sections was plotted for control, those were blocked with L-NEM and those were reduced with ascorbate and were blocked with L-NEM. This was done for both the <i>A. hypogaea</i> JL 24 (—♦— 20 day, —▪— 40 day and —▴— 80 day old nodule) and <i>M. sativa</i> (—×— 143 day old nodule). Results were expressed as mean ± SD, for n = 3 experiments.</p

Measurement of total thiol, Glutathione reductase (GR), Catalase, Ascorbate Peroxidase and Superoxide dismutase (SOD) activity in different days’ nodule extracts of <i>A. hypogaea</i>.

<p>Total thiol content was measured in 20, 40 and 80 day nodule extracts as described by Akerboom and Sies, (1981). Glutathione Reductase activity was measured spectrophotometrically at 340 nm over 2 minute by following NADPH oxidation whereas Catalase and Ascorbate Peroxidase activity was measured by following the H₂O₂ oxidation spectrophotometrically at 240 nm for 2 min in 20, 40, 80 day nodule extracts of <i>A. hypogaea.</i> SOD activity was measured in the same samples using xanthine oxidase and NBT and absorption was measured at 560 nm. Results are expressed as mean ± SD, for n = 3 experiments. P≤0.01, using one-way ANOVA.</p