Manganese (Mn) oxidation increases intracellular Mn in Pseudomonas putida GB-1.

Bacterial manganese (Mn) oxidation plays an important role in the global biogeochemical cycling of Mn and other compounds, and the diversity and prevalence of Mn oxidizers have been well established. Despite many hypotheses of why these bacteria may oxidize Mn, the physiological reasons remain elusive. Intracellular Mn levels were determined for Pseudomonas putida GB-1 grown in the presence or absence of Mn by inductively coupled plasma mass spectrometry (ICP-MS). Mn oxidizing wild type P. putida GB-1 had higher intracellular Mn than non Mn oxidizing mutants grown under the same conditions. P. putida GB-1 had a 5 fold increase in intracellular Mn compared to the non Mn oxidizing mutant P. putida GB-1-007 and a 59 fold increase in intracellular Mn compared to P. putida GB-1 ∆2665 ∆2447. The intracellular Mn is primarily associated with the less than 3 kDa fraction, suggesting it is not bound to protein. Protein oxidation levels in Mn oxidizing and non oxidizing cultures were relatively similar, yet Mn oxidation did increase survival of P. putida GB-1 when oxidatively stressed. This study is the first to link Mn oxidation to Mn homeostasis and oxidative stress protection

Protein carbonylation following oxidative stress.

<p>A) Oxyblot and B) Coomassie stained SDS-PAGE of hydrogen peroxide treated <i>P. putida</i> GB-1 grown under oxidizing (+Mn) and non-oxidizing (-Mn) conditions. Stationary phase cells were treated with 0.6 mM hydrogen peroxide for 30 minutes. The extent of protein carbonylation in cells oxidatively stressed in the presence or absence of Mn oxides was equivalent. Lane 1: negative control for non-oxidizing conditions; Lane 2,3: non-oxidizing conditions; Lane 4: Mn oxidizing negative control; Lane 5,6: Mn oxidizing conditions; Lane 7: Protein standard. The negative control was not derivatized with dinitrophenylhydrazine.</p

Survival of <i>P. putida</i> following hydrogen peroxide stress.

<p><i>P. putida</i> GB-1, GB-1-007, and ∆2665 ∆2447 strains were exposed to 16.1 mM hydrogen peroxide for 30 minutes. Relative survival was calculated as the log₁₀ of the percent survival rate of the oxidizing (+ Mn) culture divided by the percent survival rate of the non-oxidizing (-Mn) culture. The horizontal lines represent the mean value of the data sets. The mean relative survival of <i>P</i>. <i>putida</i> GB-1 is significantly higher than the mean relative survival of <i>P</i>. <i>putida</i> GB-1-007 and <i>P</i>. <i>putida</i> ∆2665 ∆2447 (P < 0.05, n=5) as determined by the Student’s t-test. Although the relative survival varied by experiment, <i>P. putida</i> GB1 consistently showed greater relative survival than the non-oxidizing mutants.</p