Effect of cell growth rate on expression of the anaerobic respiratory pathway operons frdABCD, dmsABC, and narGHJI of Escherichia coli

Abstract

The fumarate reductase (frdABCD), dimethyl sulfoxide (DMSO)-trimethylamine-N-oxide (TMAO) reductase (dmsABC), and nitrate reductase (narGHJI) operons in Escherichia coli encode enzymes involved in anaerobic respiration to the electron acceptors fumarate, DMSO or TMAO, and nitrate, respectively. They are regulated in response to anaerobiosis and nitrate availability. To determine how each operon is regulated in response to changes in cell growth rate and in oxygen availability, expression offrdA-lacZ, dmnsA-lacZ, and narG-lacZ fusion genes was examined during continuous culture. After a change in the cell growth rate, each anaerobic electron transport pathway operon fusion responded somewhat differently. WhereasfrdA-lacZ expression increased by fivefold as the growth rate decreased from 0.60 to 0.12/hour during aerobic growth, little change was seen under anaerobic conditions. In contrast, growth rate-dependent expression of narG-lacZ expression occurred under anaerobic conditions but not under aerobic conditions. Finally, dmsA-lacZ expression did not vary greatly for any of the growth rates tested. When cells were shifted from aerobic to anaerobic growth conditions, expression of each fusion increased at a moderate rate and peaked or "overshot" before reaching a new equilibrium value. This "overshoot" phenomenon was independent of the frr gene product, which functions as a transcriptional activator of each respiratory operon during anaerobic conditions. In contrast to the moderate rate of anaerobic induction seen for narG-lacZ expression, the addition of nitrate caused a rapid induction response. The cell appears to have many ways to adjust cell respiration in response to changes in cell growth conditions. Escherichia coli can respire either aerobically or anaerobically by using one of the alternative electron acceptors: oxygen, nitrate, trimethylamine-N-oxide (TMAO), dimethyl sulfoxide (DMSO), and fumarate. Depending on the availability of these respiratory substrates, the cell synthesizes one or more of the terminal enzymes of the electron transport pathways. DMSO-TMAO reductase, encoded by dmsABC, exhibits a broad substrate specificity for reducing TMAO, DMSO, and other amine-N-oxides (4, 28). Fumarate reductase readily catalyzes the interconversion of fumarate and succinate, although under physiological conditions, it is thought to perform the reductive reaction primarily during anaerobic growth (1). The four enzyme subunits are encoded by the frdABCD genes. The inducible nitrate reductase enzyme complex of E. coli which converts nitrate to nitrite during anaerobic cell growth is encoded by the narGHJI genes (13,27). The frdABCD, dmsABC, and narGHJI operons that encode the subunits of each enzyme complex are unlinked to one another and map at 94, 20, and 27 min on the E. coli chromosome, respectively (2).Expression of the anaerobic electron transport pathway operons is regulated in response to anaerobiosis and by the availability of nitrate (17, 18). The anaerobic response is mediated by the fnr gene product...

Effect of microaerophilic cell growth conditions on expression of the aerobic (cyoABCDE and cydAB) and anaerobic (narGHJI, frdABCD, and dmsABC) respiratory pathway genes in Escherichia coli

“…A modified Vogel-Bonner medium (pH 6.5) supplemented with Casamino Acids (0.25 mg/liter) and glucose (2.25 mM) was used to limit cell growth (i.e., carbon-limited medium). Aerobic continuous culture conditions were maintained by saturating the culture medium with sterile air at a flow rate of 200 ml/min, and anaerobic conditions were maintained by continuously sparging the vessel with oxygen-free nitrogen at a flow rate of 200 ml/min (19). To vary the degree of air saturation of the medium, the vessel was sparged with a stream of premixed gas in which the proportion of compressed air (21% O 2 ) and compressed nitrogen (99.98%) was controlled by using a manifold with precalibrated flow meters for each gas.…”

“…For continuous-culture experiments, a Mouse bioreactor (Queue Corp., Parkersburg, W.V.) was fitted with a 2-liter vessel and operated at a 1-liter liquid working volume as previously described (19). A modified Vogel-Bonner medium (pH 6.5) supplemented with Casamino Acids (0.25 mg/liter) and glucose (2.25 mM) was used to limit cell growth (i.e., carbon-limited medium).…”

“…To ensure that no deleterious mutations had occurred in the lacZ indicator strain, cell samples were taken from the chemostat periodically and grown in batch culture for ␤-galactosidase assays to confirm that the expression of each lacZ fusion was wild type. Minimal glucose-X-Gal plates (19) for strain testing were prepared with Difco Bacto Agar (15 g/liter) and X-Gal (40 g/ml).…”

Membrane Potential as a Regulation Mechanism of Periplasmic Nitrite Reductase Activity: A Mathematical Model

“…Показано также, что основным ферментом, потребляющим Н 2 в анаэробных условиях, является Hyd-2 гидрогеназа [18]. Учитывая, что клетки E. coli в хемостате культивируются при слабокислом рН = 6.5 и низкой концентрации глюкозы в среде (2.25 мМ) [20], можно предположить наличие активности FHL-1 комплекса в условиях стационарного роста клеток в хемостате. Исходя из этого, были предложены следующие гипотетические сценарии формирования респираторной цепи с участием FHL-1 комплекса в условиях культивирования клеток E. coli в хемостате и при низких концентрациях нитрита в среде (<1 мМ).…”

“…Эта возможность просматривается в структуре HycD/HycC субъединиц Hyd-3 фермента, обладающих значительным сходством с респираторным комплексом НАДН:хиноноксидоредуктазы (Complex 1) [27,16,28], и продемонстрирована в экспериментах у двойных мутантов c делециями оперонов hya [Hyd-1] и hyb [13], проведенных при рН = 6.5 в ферментирующих условиях, что совпадает с условиями роста клеток в проточном хемостате [20] в отсутствие нитрита. В случае прямой транслокации протонов через FHL-1 комплекс, механизм формирования мембранного потенциала будет идентичен таковому с участием FHL-2 комплекса, который рассматривается далее.…”