Relationship between Succinate Transport and Production of Extracellular Poly(3-Hydroxybutyrate) Depolymerase in Pseudomonas lemoignei

Abstract

The relationship between extracellular poly(3-hydroxybutyrate) (PHB) depolymerase synthesis and the unusual properties of a succinate uptake system was investigated in Pseudomonas lemoignei. Growth on and uptake of succinate were highly pH dependent, with optima at pH 5.6. Above pH 7, growth on and uptake of succinate were strongly reduced with concomitant derepression of PHB depolymerase synthesis. The specific succinate uptake rates were saturable by high concentrations of succinate, and maximal transport rates of 110 nmol/mg of cell protein per min were determined between pH 5.6 and 6.8. The apparent K S0.5 values increased with increasing pH from 0.2 mM succinate at pH 5.6 to more than 10 mM succinate at pH 7.6. The uptake of [14C]succinate was strongly inhibited by several monocarboxylates. Dicarboxylates also inhibited the uptake of succinate but only at pH values near the dissociation constant of the second carboxylate function (pKa2). We conclude that the succinate carrier is specific for the monocarboxylate forms of various carboxylic acids and is not able to utilize the dicarboxylic forms. The inability to take up succinate2− accounts for the carbon starvation of P. lemoignei observed during growth on succinate at pH values above 7. As a consequence the bacteria produce high levels of extracellular PHB depolymerase activity in an effort to escape carbon starvation by utilization of PHB hydrolysis products.

Poly(3-hydroxybutyrate)-depolymerase from Pseudomonas lemoignei: Catalysis of Esterifications in Organic Media

“…Preparation and Assay of PHB-depolymerase. P. lemoignei was grown in 10 L of Stinson and Merrick's mineral medium with 50 mM sodium succinate in a Braun Biostat fermentor (model ES 10) at 30 °C for 24 h. The conditions of maximal PHB-depolymerse production by P. lemoignei have recently been described . The cell-free culture fluid was obtained by centrifugation, and the proteins were concentrated 10-fold by ultrafiltration (exclusion size 30 kDa).…”

The “PHB Depolymerase Inhibitor” of Paucimonas lemoignei Is a PHB Depolymerase

“…P. lemoignei produces high amounts of PHB depolymerase isoenzymes during growth on succinate in batch culture. 6 Recently, we had isolated a new PHB depolymerase, PhaZ7, from succinate-grown culture fluid. 5 It came out that the purified new depolymerase had an apparent molecular mass of 36 kDa.…”

“…The relationship between expression of PHB depolymerase activity and growth on succinate has been studied earlier. 6 However, knowledge of the regulation of PHB depolymerase synthesis on the molecular level is still low. A few years ago Doi and co-workers described a 35 kDa protein that was secreted from P. lemoignei during growth on succinate and that inhibited the activity of dPHB depolymerases in vitro (PHB depolymerase inhibitor, PDI).…”

“…In contrast to most PHB-degrading bacteria, which repress PHB depolymerase synthesis in the presence of soluble carbon sources such as glucose or fatty acids, P. lemoignei synthesizes high PHB depolymerases activity during growth on succinate. The relationship between expression of PHB depolymerase activity and growth on succinate has been studied earlier . However, knowledge of the regulation of PHB depolymerase synthesis on the molecular level is still low.…”

Molekularbiologische und biochemische Untersuchungen zum bakteriellen Naturkautschuk-Abbau, sowie Charakterisierung eines dazu befähigten Bakteriums

“…Vorhandensein des lipophilen Proteinanteils bedeutet bei Gram-positiven Bakterien nicht zwangsläufig eine Bindung an die Zelloberfläche, wurden doch viele Lipoproteine Gram-positiver Bakterien aus dem Kulturüberstand aufgereinigt (Rothe et al, 1989;Sutcliffe et al, 1993) , b , , c , (Linos & Steinbüchel, 1998), d , (Linos et al, 1999), e , (Rook, 1955), f , , g , ), h , (Jendrossek et al, 1997) (Priest, 1992). Beim Wachstum auf Succinat und pH-Werten ≥ 6,8 werden die PHB-Depolymerasen aus Pseudomonas lemoignei exprimiert und sekretiert, während die Anwesenheit von 3-Hydroxybutyrat diese Schritte unterdrückt (Delafield et al, 1965;Terpe, 1999). Auch die Laccase-Produktion des…”