Cytochrome bd Oxidase, Oxidative Stress, and Dioxygen Tolerance of the Strictly Anaerobic Bacterium Moorella thermoacetica

Abstract

The gram-positive, thermophilic, acetogenic bacterium Moorella thermoacetica can reduce CO 2 to acetate via the Wood-Ljungdahl (acetyl coenzyme A synthesis) pathway. This report demonstrates that, despite its classification as a strict anaerobe, M. thermoacetica contains a membrane-bound cytochrome bd oxidase that can catalyze reduction of low levels of dioxygen. Whole-cell suspensions of M. thermoacetica had significant endogenous O 2 uptake activity, and this activity was increased in the presence of methanol or CO, which are substrates in the Wood-Ljungdahl pathway. Cyanide and azide strongly (ϳ70%) inhibited both the endogenous and CO/methanol-dependent O 2 uptake. UV-visible light absorption and electron paramagnetic resonance spectra of n-dodecyl-␤-maltoside extracts of M. thermoacetica membranes showed the presence of a cytochrome bd oxidase complex containing cytochrome b 561 , cytochrome b 595 , and cytochrome d (chlorin). Subunits I and II of the bd oxidase were identified by N-terminal amino acid sequencing. The M. thermoacetica cytochrome bd oxidase exhibited cyanide-sensitive quinol oxidase activity. The M. thermoacetica cytochrome bd (cyd) operon consists of four genes, encoding subunits I and II along with two ABC-type transporter proteins, homologs of which in other bacteria are required for assembly of the bd complex. The level of this cyd operon transcript was significantly increased when M. thermoacetica was grown in the absence of added reducing agent (cysteine ؉ H 2 S). Expression of a 35-kDa cytosolic protein, identified as a cysteine synthase (CysK), was also induced by the nonreducing growth conditions. The combined evidence indicates that cytochrome bd oxidase and cysteine synthase protect against oxidative stress and contribute to the limited dioxygen tolerance of M. thermoacetica.Dioxygen is the preferred electron sink in the respiratory chain of most aerobic bacteria (58). Cytochrome oxidases are the membrane-bound, terminal components of the dioxygendependent respiratory chain, which reduces dioxygen to water with formation of pH and potential gradients (6, 45). Two main types of respiratory cytochrome oxidases are known in bacteria: the heme/copper oxidases (cytochrome aa 3 , cytochrome caa 3 oxidase, and cytochrome bo oxidase), and the heme-only cytochrome bd quinol oxidase, which is associated with microaerobic dioxygen respiration (24,56,59,62). Cytochrome bd oxidases purified from aerobic bacteria have been characterized as 1:1 heterodimers of two integral membrane proteins referred to as subunits I and II (35,55). The heterodimers contain three heme components: a low-spin heme, b 558 , and two high-spin hemes, b 595 and d. In the genomes of Escherichia coli (31) and Azotobacter vinelandii (41), subunits I and II are encoded by cydA and cydB, respectively, of the cytochrome bd (cyd) operon. Two ABC-type transporter proteins, CydD and CydC, are typically required for assembly of the cytochrome bd complex in both gram-positive and gram-negative aerobic bacteria (14, 60). Aerobic r...

A Life with Acetogens, Thermophiles, and Cellulolytic Anaerobes

“…Thus, it seemed warranted to isolate sugar phosphates from the cells of M. thermoacetica after exposure to 14 CO 2 and to determine the distribution of radioactivity in them. Another possible mechanism relates to the isotope exchange between 14 CO 2 and formate as catalyzed by resting cells of M. thermoacetica, and to the finding by Kenneth Lentz (37) that formate may be a better precursor of the methyl group of acetate.…”

“…After a few days it was time for me to work and to learn about the use of radioactive isotopes and how to degrade biological compounds to determine the incorporation and distribution of 14 C-labeled precursors in these compounds. At the time, Wood was working on two projects.…”

“…He had already found that a substantial randomization of the 14 C glucose occurred, and he wanted to establish the mechanism of that as well as find a method to calculate the involvement of the pentose cycle (84). My job was to purify sugar phosphates and, after dephosphorylation, determine the distribution of 14 C among the carbons. One study I was involved with was determining whether transaldolase catalyzes an exchange reaction between glyceraldehyde-3-phosphate and fructose-6-phosphate.…”

“…There were still problems with the pathway involving the formation of propionic acid in Propionibacterium shermanii, the bacterium with which Wood had established heterotrophic CO 2 fixation. The second project was studying the metabolism of 14 C glucose in mammalian tissues, especially lactose synthesis in the mammary gland. He had already found that a substantial randomization of the 14 C glucose occurred, and he wanted to establish the mechanism of that as well as find a method to calculate the involvement of the pentose cycle (84).…”

Trophic interaction of the aerotolerant anaerobe Clostridium intestinale and the acetogen Sporomusa rhizae sp. nov. isolated from roots of the black needlerush Juncus roemerianus

“…Acetogens possess a variety of oxidoreducatases that can reduce O 2 and its toxic by-products peroxide and superoxide. For example, the model acetogen M. thermoacetica contains peroxidase, NADH oxidase, rubredoxin oxidoreductase (a superoxide reductase) and rubrerythrin (a peroxidase), and also has the ability to dissimilate nitrate to ammonium (a more redoxpositive half-cell reaction than that of CO 2 /acetate) as an alternative terminal electron-accepting process (Das et al, 2001(Das et al, , 2005Karnholz et al, 2002;Silaghi-Dumitrescu et al, 2003;Drake & Daniel, 2004). Marine and estuarine plants grow in sediments that are constantly or frequently water saturated, and anoxia can occur at depths of only a few millimetres in flooded sediments.…”