Citrate metabolism in <i>Pseudomonas fluorescens</i> under nitrosative stress.

<p>Increased activity and expression of CL allows for the degradation of citrate and circumvention of nitrosative stress via a novel metabolic network in <i>P. fluorescens</i> subjected to SNP (⇓, decreased activity, ⇑, increased activity). CL, citrate lyase; AK, acetate kinase; ICDH, isocitrate dehydrogenase; MDH, malate dehydrogenase; PEPC, phosphoenolpyruvate carboxylase; PPDK, pyruvate phosphate dikinase; PDH, pyruvate dehydrogenase.</p

Oxaloacetate and PEP metabolism.

<p><i>A</i>, BN-PAGE analysis of oxaloacetate metabolizing enzymes. Bands were quantified using SCION Imaging Software (n = 3± standard deviation). <i>B</i>, In-cell Western blot analysis. <i>P.fluorescens</i> was grown to late logarithmic phase in 96 well plates then treated with control (−) and 10 mM SNP conditions for 24 h (+SNP). The cells were then analyzed for <b>(</b>GDH) and (PEPC) and expression was quantified by infrared fluorescence (n = 5, mean ± standard deviation. *p≤0.01). C, Citrate consumption in <i>P. fluorescens</i> exposed to ntirosative stress. Soluble CFE from 10 mM SNP-treated cells was incubated with 2 mM citrate, 1.0 mM Pi and 0.5 mM AMP for 30 min without (▪) and with an antibody (1∶500 dilution) against PEPC (□). n = 3, mean ± standard deviation. D, in-gel activity stain of the pyruvate generating enzymes PK and PPDK (−, control cultures; <i>+</i>SNP, 10 mM SNP-treated cultures).</p

Activity and expression analysis of CL from <i>P. fluorescens</i>.

<p><i>A,</i> in-gel CL activity in soluble CFE isolated from <i>P. fluorescens</i>. <b>I</b>) The dose effect of SNP on CL activity <b>II</b>) Time dependence of CL activity in cultures treated with 10 mM SNP. <b>III</b>) CL activity in media containing 1 mM DEANO (−, control cultures; +DEANO, 1 mM DEANO-treated cultures). <i>B</i>, 2D SDS-PAGE (−, control cultures; <i>+</i>SNP, 10 mM SNP-treated cultures). <i>C</i>, regulation of CL activity by SNP. 10 mM SNP-treated cells were grown to late logarithmic phase then transferred to control media for 8 h and vice versa. <i>D</i>, HPLC analysis of CL activity. Soluble CFE from cells grown in control and 10 mM SNP-treated media were given 5 mM citrate and 0.5 mM NADP for 2 hrs (*: tricarballylic acid (1 µM) as internal standard).</p

Influence of NO on the TCA cycle.

<p><i>A,</i> Representative chromatographs showing metabolite levels in soluble CFE from <i>P. fluorescens</i> grown in control and 10 mM SNP-containing cultures. <i>B,</i> In-gel activity analysis for ACN. <i>C</i>, in-gel activity staining of key metabolic enzymes. <i>D</i>, activity stains with sodium ferrocyanide as a control (−, control cultures; <i>+</i>SNP, 10 mM SNP-treated cultures; +SFC, 10 mM SFC-treated cultures).</p

RNS detoxifying mechanisms in <i>P. fluorescens</i>.

<p><i>A</i>, BN-PAGE analysis of NIR and NR from membrane CFE: ▪, control cultures; ▪, 10 mM SNP-treated cultures. Bands were quantified using SCION Imaging Software (n = 3, mean ± standard deviation). <i>B</i>, activity staining of GSNOR in soluble CFE (−, control cultures; <i>+</i>SNP, 10 mM SNP-treated cultures).</p

Nitrosative stress in <i>P. fluorescens</i>.

<p><i>A,</i> cellular yield: ▴, control cultures; ♦, 5 mM SNP-containing cultures; •, 10 mM SNP-containing cultures; ▪, 15 mM SNP-containing cultures; □, 20 mM SNP-containing cultures. Nitrite/nitrate levels in spent fluids as measured by their absorbance at 540 nm after the Griess assay: □, control cultures; ▪, 5 mM SNP-containing cultures; ▪, 10 mM SNP-containing cultures. <i>B</i>, In cell Western blot analysis of <i>P. fluorescens</i> under nitrosative stress. <i>P. fluorescens</i> was grown to late logarithmic phase in 96 well plates and then treated with control (−) and 10 mM SNP conditions for 24 h (+SNP). Following treatment, control cells were placed in media containing 10 mM SNP for 8 h (Ctl→SNP) and 10 mM SNP-treated cells were recovered with control media for 8 h (SNP→Ctl). Total protein concentration was assessed by the Bradford assay. The cells were then analyzed for expression of nitrosylated tyrosine residues by quantification of infrared fluorescence (n = 3. *p≤0.01, mean ± standard deviation).</p