O-Acetyl-L-serine sulfhydrylase (EC 4.2.99.8) was first purified from an extremely thermophilic bacterium, Thermus thermophilus HB8, in order to ascertain that it is responsible for the cysteine ...synthesis in this organism cultured with either sulfate or methionine given as a sole sulfur source. Polyacrylamide gel electrophoreses both with and without SDS found high purity of the enzyme preparations finally obtained, through ammonium sulfate fractionation, ion exchange chromatography, gel filtration, and hydrophobic chromatography (or affinity chromatography). The enzyme activity formed only one elution curve in each of the four different chromatographies, strongly suggesting the presence of only one enzyme species in this organism. Molecular massesof 34,000 and 68,000 were estimated for dissociated subunit and the native enzyme, respectively, suggesting a homodimeric structure. The enzyme was stable at 70degC at pH 7.8 for 60 min, and more than 90% of the activity was retained after incubation of its solution at 80degC with 10 mM dithiothreitol. The enzyme was also quite stable at pH 8-12 (50degC, 30 min). It had an apparent Km of 4.8 mM for O-acetyl-L-serine (with 1 mM sulfide) and a Vmax of 435micro mol/min/mg of protein. The apparent Km for sulfide was approximately 50micro M (with 20 mm acetylserine), suggesting that the enzyme can react with sulfide liberated very slowly from methionine. The absorption spectrum of the holo-enzyme and inhibition of the activity by carbonyl reagents suggested the presence of pyridoxal 5'-phosphate as a cofactor. The apo-enzyme showed an apparent Km of 29micro M for the cof actor at pH 8. Monoiodoacetic acid (1 mM) almost completely inactivated the enzyme. The meaning of a very high enzyme content in the cell is discussed.
To gain insight into the molecular aspects of unfolding/refolding of enzymes from extremophilic organisms, we have used xylanase from an alkalophilic thermophilic Bacillus as the model system. ...Kinetics of denaturation/renaturation were monitored using intrinsic fluorescence studies. The protein fluorescence measurements suggested a putative intermediate state present in 0.08 M guanidine hydrochloride with an emission maximum of 345 nm; the far-UV circular dichroism spectra revealed content of secondary structure similar to the native enzyme. Studies with the fluorescent apolar probe 1-anilinonapthalene-8-sulfonate (1,8-ANS) were consistent with the presence of increased hydrophobic surfaces as compared with the native or fully unfolded protein. The refolding of Xyl II, was attempted by a relatively new strategy using an artificial chaperone assisted two-step method. The unfolded xylanase was found to bind to the detergent transiently and the subsequent addition of methyl-beta-cyclodextrin helped to strip the detergent and assist in the folding. Our findings suggested that the detergent stabilized a putative intermediate in the folding pathway seemingly equivalent to the folding state described as molten globule. The reactivation of Xyl II was affected by ionic as well as nonionic detergents. However, the cationic detergent cetyltrimethylammonium bromide (CTAB) provided a maximum reactivation (threefold) of the enzyme. The 'delayed detergent addition' experiments revealed that the detergent acts by suppressing the initial aggregate formation and not by dissolving aggregates. The relevance of our findings to the role of artificial chaperones in vivo is discussed.
The complete genomic DNA sequence of the thermophilic archaeon Thermoplasma volcanium is visually represented by assembling micro-squares in four color-types. The number of bases in this sequence ...(1,584,804) is larger than what was reported earlier (1,584,799 in Kawashima, T., Yamamoto, Y., Aramaki, H., Nunoshiba, T., Kawamoto, T. et al., 1999, Proc. Japan Acad. 75B, 213-218) by 5. These five bases (A at position 320,375, C at 342,507, T at 1,182,809, A at 1,365,060, and C at 1,376,292 in the new version) are missing from the previous version, mainly, not because of erroneous sequence reading, but due to physical deletion of corresponding base-pairs upon replication of the cloned DNA fragments in E. coli cells. (Communicated by Masanori OTSUKA, M. J. A., Jan. 14, 2003)
To gain insight into the molecular aspects of unfolding/refolding of enzymes from extremophilic organisms, we have used xylanase from an alkalophilic thermophilic Bacillus as the model system. ...Kinetics of denaturation/renaturation were monitored using intrinsic fluorescence studies. The protein fluorescence measurements suggested a putative intermediate state present in 0.08 m guanidine hydrochloride with an emission maximum of 345 nm; the far‐UV circular dichroism spectra revealed content of secondary structure similar to the native enzyme. Studies with the fluorescent apolar probe 1‐anilinonapthalene‐8‐sulfonate (1,8‐ANS) were consistent with the presence of increased hydrophobic surfaces as compared with the native or fully unfolded protein. The refolding of Xyl II, was attempted by a relatively new strategy using an artificial chaperone assisted two‐step method. The unfolded xylanase was found to bind to the detergent transiently and the subsequent addition of methyl‐β‐cyclodextrin helped to strip the detergent and assist in the folding. Our findings suggested that the detergent stabilized a putative intermediate in the folding pathway seemingly equivalent to the folding state described as molten globule. The reactivation of Xyl II was affected by ionic as well as nonionic detergents. However, the cationic detergent cetyltrimethylammonium bromide (CTAB) provided a maximum reactivation (threefold) of the enzyme. The ‘delayed detergent addition’ experiments revealed that the detergent acts by suppressing the initial aggregate formation and not by dissolving aggregates. The relevance of our findings to the role of artificial chaperones in vivo is discussed.
This article describes how geological processes underlie all environments in which microbes thrive. Some of these environments favor the transmission of pathogens to humans, and others can be altered ...to do so by anthropogenic processes or broader ecological disruptions. We provide examples of each including: extremophile microbes (able to thrive in almost unbelievably harsh environmental conditions); microbes in natural environments that can opportunistically infect people coming into contact therewith; pathogens that emerge from environments heavily modified by human activity such as agriculture; and pathogens that emerge as major public health problems because of large-scale ecological disruption. Finally, future scenarios with a low probability but high potential impact are considered, such as the introduction to Earth’s ecosystems of microbes that have evolved in extraterrestrial geological environments.
Nanoarchaeota St. John, Emily; Reysenbach, Anna-Louise
Encyclopedia of Microbiology,
2019
Book Chapter
The Nanoarchaeota are a group of symbiotic Archaea that engage in close interspecies associations with diverse archaeal hosts. Nanoarchaeote sequences have been recovered from high-temperature ...geothermal springs and marine hydrothermal vents around the world. However, few Nanoarchaeota have been successfully isolated with their hosts in the laboratory. Cultivated nanoarchaeotes are ectosymbionts with small cell diameters (~100–400 nm) and reduced genomes (0.491–0.606 Mbp). Described Nanoarchaeota lack most genes involved in major biosynthetic pathways and likely obtain many cellular products directly from their hosts. Without a complete ATP synthase complex, nanoarchaeotes may also rely on their hosts to provide ATP. Described nanoarchaeote genomes show varying levels of reduction, consistent with a host-reliant lifestyle. However, some nanoarchaeotes retain genes involved in the gluconeogenesis pathway, archaeal flagella and CRISPR-Cas systems.
Organisms from extreme environments provide hearty enzymatic and whole-cell systems for biocatalysis. These intriguing systems hold possibilities in situations that inhibit reactions for typical ...bioconversion systems. Scientists and engineers have expanded their understanding of the systems that control catalysis under extremes and have insights into the stability and adaptability of extreme systems. Through examining selective pressures and identifying extremophilic systems that survive and evolve in contaminated environments, it is possible to engineer novel remediation applications through making extremophiles work on a wider range of substrates or by engineering more stability into mesophilic systems. This chapter explores recent advances in extremophilic bioremediation and provides a survey of enzymatic and whole-cell applications.
Physiological effects of NaCl concentration (equivalent water activities, a^sub w^, 1 to 0.87) were investigated with the moderately halophilic and piezotolerant bacterium, Kocuria rosea (formerly ...Micrococcus roseus), grown in bacteriological peptone/yeast extract broth. This bacterium, which was isolated from open shallow seawater, can grow in 150 g NaCl l^sup -1^ (optimum NaCl concentration: 30 g l^sup -1^, a^sub w^=0.984) and under 207 MPa of hydrostatic pressure. The effects of water activity on μ^sub m^ can be quantitatively predicted, to a high level of accuracy by application of the Aiba/Edwards, and the Levenspiel-type unstructured inhibition-type kinetic models.PUBLICATION ABSTRACT
Extremophilic organisms are adapted to harsh environmental conditions like high temperature, extremely acidic or alkaline pH, high salt, or a combination of those. With a few exceptions extremophilic ...bacteria are colonizing only moderately hot biotopes, whereas hyperthermophiles are found specifically among archaea (formerly ‘archaebacteria’) which can thrive at temperatures close to or even above the boiling point of water 1. It has been a challenging question whether the special properties of their proteins and membranes have been acquired by adaptation, or whether they might reflect early evolutionary states as suggested by their phylogenetic position at the lowest branches of the universal tree of life 2.
Abstract
We have recovered new isolates from hot springs, in Yellowstone National Park and the Kamchatka Peninsula, after γ-irradiation and exposure to high vacuum (10−6 Pa) of the water and sediment ...samples. The resistance to desiccation and ionizing radiation of one of the isolates, Bacillus sp. strain PS3D, was compared to that of the mesophilic bacterium, Deinococcus radiodurans, a species well known for its extraordinary resistance to desiccation and high doses of ionizing radiation. Survival of these two microorganisms was determined in real and simulated space conditions, including exposure to extreme UV radiation (10–100 nm) during a rocket flight. We found that up to 15 days of desiccation alone had little effect on the viability of either bacterium. In contrast, exposure to space vacuum (∼10−6 Pa) decreased cell survival by two and four orders of magnitude for Bacillus sp. strain PS3D and D. radiodurans, respectively. Simultaneous exposure to space vacuum and extreme UV radiation further decreased the survival of both organisms, compared to unirradiated controls. This is the first report on the isolated effect of extreme UV at 30 nm on cell survival. Extreme UV can only be transmitted through high vacuum, therefore its penetration into the cells may only be superficial, suggesting that in contrast to near UV, membrane proteins rather than DNA were damaged by the radiation.
PDF
