Fermentative and aerobic metabolism in Rhizobium etli

Abstract

Strains of Rhizobium etli, Rhizobium meliloti, and Rhizobium tropici decreased their capacity to grow after successive subcultures in minimal medium, with a pattern characteristic for each species. During the growth of R. etli CE 3 in minimal medium (MM), a fermentation-like response was apparent: the O 2 content was reduced and, simultaneously, organic acids and amino acids were excreted and poly-␤-hydroxybutyrate (PHB) was accumulated. Some of the organic acids excreted into the medium were tricarboxylic acid (TCA) cycle intermediates, and, concomitantly, the activities of several TCA cycle and auxiliary enzymes decreased substantially or became undetectable. Optimal and sustained growth and a low PHB content were found in R. etli CE 3 when it was grown in MM inoculated at a low cell density with O 2 maintained at 20% or with the addition of supplements that have an effect on the supply of substrates for the TCA cycle. In the presence of supplements such as biotin or thiamine, no amino acids were excreted and the organic acids already excreted into the medium were later reutilized. Levels of enzyme activities in cells from supplemented cultures indicated that carbon flux through the TCA cycle was maintained, which did not happen in MM. It is proposed that the fermentative state in Rhizobium species is triggered by a cell density signal that results in the regulation of some of the enzymes responsible for the flux of carbon through the TCA cycle and that this in turn determines how much carbon is available for the synthesis and accumulation of PHB. The fermentative state of free-living Rhizobium species may be closely related to the metabolism that these bacteria express during symbiosis.In aerobic bacteria, the tricarboxylic acid (TCA) cycle functions to generate reduced nucleotides by the complete oxidation of pyruvate, which enters the cycle in the form of acetyl coenzyme A (acetyl-CoA). The reduced nucleotides are then used to generate ATP via the electron transport system. Another major function is to produce intermediates for anabolism, and several anaplerotic reactions serve to replenish TCA cycle intermediates which are consumed in these processes (32).The accumulation of the microbial reserve polyester poly-␤-hydroxybutyrate (PHB) in bacteria is well documented (1,6,45), as is the presence of PHB in several species of Rhizobium, both in the free state (44, 46) and in symbiosis (14,19,48). While several different pathways for the production of PHB in various groups of bacteria have been characterized (1), the most common pathway begins with the condensation of two molecules of acetyl-CoA to form acetoacetyl-CoA. Sequential reduction and polymerization reactions produce PHB. This product can be depolymerized and ultimately converted back to acetyl-CoA (Fig. 1). Like the TCA cycle, carbon flux through this pathway is greatly influenced by growth conditions, and the two cycles must compete for a common starting metabolite, acetyl-CoA. However, the function of PHB in cell metabolism in Rhizobium species has...

Production of Poly-β-hydroxybutyric acid (PHB) by Bacillus cereus

“…The PHB yields increased with time dependent manner and highest yield 3.6 g/L was obtained after 48 h for growth of B. cereus. The PHB yield decreased to 2.1 at 72 h. According to Bonartseva et al, (1994) the results confirmed the observed maximal PHB accumulation at 48 h. After 48 h there was a decrease in PHB yield and an increase in the viscosity of the medium at 72, 96 and 120 h. After 48 h, then unfavorable conditions of the medium caused the decrease in PHB yield, because the increase in medium viscosity accompany exopolysaccharides production resulting in oxygen transfer limitation, caused the decrease in PHB synthesis, ( Stam et al, 1986a;Tombolini and Nuti, 1989;Encarnacion et al, 1995). The yield decreased at 72 and 120 h although dry cell weight increased at 120 h, the decrease of PHB indicates that the bacteria used PHB as a source of carbon, causing unsuitable conditions due to inadequate nitrogen and carbon sources in the medium.…”

Plasmids with a Chromosome-Like Role in Rhizobia

“…To prevent growth arrest by metabolic imbalance in subsequent cultures, MMY was also supplemented with biotin at 1 mg liter Ϫ1 (19). When needed, amino acids or vitamins were added to MMY at the following concentrations (in g ml Ϫ1 ): cobalamin, 0.5; methionine, 100; and nicotinic acid, 12.…”

Regulatory proteins and cis-acting elements involved in the transcriptional control of Rhizobium etli reiterated nifH genes

“…Plasmids and strains used in this study are described in Table 1. Rhizobium strains were grown at 30ЊC in PY (peptoneyeast) complex medium or minimal medium containing succinate and ammonium chloride as the sole carbon and nitrogen sources (18). Escherichia coli strains were grown at 37ЊC in Luria-Bertani complex medium or at 30ЊC in minimal medium containing 10 mM glucose, 10 mM ammonium chloride or glutamine as the nitrogen source, 11 mM K 2 HPO 4 , 51 mM KH 2 PO 4 , and 4 mM MgSO 4 ⅐ 7H 2 O.…”