Biochemical Properties and Regulated Gene Expression of the Superoxide Dismutase from the Facultatively Aerobic Hyperthermophile Pyrobaculum calidifontis

Abstract

Superoxide dismutase (SOD) was purified from a facultatively aerobic hyperthermophilic archaeon, Pyrobaculum calidifontis VA1. The purified native protein from aerobically grown cells exhibited 1,960 U of SOD activity/mg and contained 0.86 ؎ 0.04 manganese and <0.01 iron atoms per subunit. The gene encoding SOD was cloned and expressed in Escherichia coli. Although the recombinant protein was soluble, little activity was observed due to the lack of metal incorporation. Reconstitution of the enzyme by heat treatment with either Mn or Fe yielded a highly active protein with specific activities of 1,970 and 434 U/mg, respectively. This indicated that the SOD from P. calidifontis was a cambialistic SOD with a preference toward Mn in terms of activity. Interestingly, reconstitution experiments in vitro indicated a higher tendency of the enzyme to incorporate Fe than Mn. When P. calidifontis was grown under anaerobic conditions, a majority of the native SOD was incorporated with Fe, indicating the cambialistic property of this enzyme in vivo. We further examined the expression levels of SOD and a previously characterized Mn catalase from this strain in the presence or absence of oxygen. Northern blot, Western blot, and activity measurement analyses revealed that both genes are expressed at much higher levels under aerobic conditions. We also detected a rapid response in the biosynthesis of these enzymes once the cells were exposed to oxygen.

Transcriptional Map of Respiratory Versatility in the Hyperthermophilic Crenarchaeon Pyrobaculum aerophilum

“…P. aerophilum IM2 cultures derived from DSMZ strain 7523 were generously provided by Christopher House, Penn State University. Cultures of P. aerophilum were grown in media containing (per liter) 10 g NaCl, 1.3 g (NH 4 ) 2 The complete medium preparation was adjusted to pH 6.8 and sterilized by autoclaving.…”

“…The complete genome sequence of P. aerophilum was published in 2002 (23). The genome sequences of four additional Pyrobaculum species (P. arsenaticum, P. calidifontis, P. islandicum, and Thermoproteus neutrophilus [to be reclassified as a true Pyrobaculum]) have recently been completed (T. M. Lowe et al, unpublished data), providing a valuable resource for comparing gene content to respiratory characteristics within a closely related group of crenarchaea (Table 1).A number of Pyrobaculum gene products have been characterized by heterologous expression or purification of native proteins, helping to identify genes for aerobic growth, oxidative stress, and denitrification (1,4,5,18,27,44,66). Although Pyrobaculum species are among the best-studied archaeal iron respirers, and are the only known archaeal arsenate respirers, the genes required for archaeal arsenate and iron respiration have not been identified (20,21,29,31).…”

“…A number of Pyrobaculum gene products have been characterized by heterologous expression or purification of native proteins, helping to identify genes for aerobic growth, oxidative stress, and denitrification (1,4,5,18,27,44,66). Although Pyrobaculum species are among the best-studied archaeal iron respirers, and are the only known archaeal arsenate respirers, the genes required for archaeal arsenate and iron respiration have not been identified (20,21,29,31).…”

Cloning, expression, and characterization of thermostable Manganese superoxide dismutase from Thermoascus aurantiacus var. levisporus

“…In industry, a major requirement for commercial SODs is thermal stability, because thermal denaturation is a common cause of enzyme inactivation. It has been reported that SODs were isolated from hyperthermophiles of the genera Sulfolobus, Aquifex, Pyrophilus, Pyrobaculum, and Aeropyrum (Lim et al, 1997;Knapp et al, 1999;Amo et al, 2003). The primers for PCR used in the study Primer name Sequence (5'-3') Purpose…”

Specificity of Fur Binding to the Oxidative Stress Response Gene Promoter in the Facultative Anaerobic Archaeon <i>Thermoplasma volcanium</i>

“…18) In Escherichia coli, Fur down-regulates the sodA gene, which encodes Mn-superoxide dismutase, but up-regulates sodB, which encodes Fe-superoxide dismutase, via re- pression of the small RNA RyhB, leading to rapid degradation of the sodB transcript. 19) Several bacterial Fur deletion mutant strains are known to be sensitive to ROS.…”