THERMOACIDOPHILIC BACTERIA

SUMMARY

The extremely thermoacidophilic archaea are a particularly intriguing group of microorganisms that must simultaneously cope with biologically extreme pHs (≤ 4) and temperatures (Topt ≥ 60°C) in their natural environments. Their expandi ng biotechnological significance relates to their role in biomining of base and precious metals and their unique mechanisms of survival in hot acid, at both the cellular and biomolecular levels. Recent developments, such as advances in understanding of heavy metal tolerance mechanisms, implementation of a genetic system, and discovery of a new carbon fixation pathway, have been facilitated by availability of genome sequence data and molecular genetic systems. As a result, new insights into the metabolic pathways and physiological features that define extreme thermoacidophily have been obtained, in some cases suggesting prospects for biotechnological opportunities.

Keywords: extreme thermoacidophile, bioleaching, CO2 fixation, archaeal genetics

INTRODUCTION

Over the past 20 years much has been written about the biotechnological potential of microorganisms from extreme environments, primarily focusing on individual enzymes capable of withstanding the otherwise harsh conditions required for long-term efficacy in bioprocessing environments. However, as genome sequence data have become available for extremophiles (The UCSC Archaeal Genome Browser; and molecular genetics tools have begun to emerge, there exists the possibility to go beyond single biocatalytic steps to take advantage of the novel pathways and physiological characteristics that are intrinsic to these unique microorganisms. By incorporating these features into less extreme organisms and cells and by metabolically engineering extremophiles directly, a new horizon in microbial biotechnology can emerge. Here, we consider the extremely thermoacidophilic archaea, microorganisms that thrive in hot acid.

Extremely thermoacidophilic archaea and their physiological characteristics

For the purposes of this review, an “extreme thermoacidophile” is a microorganism with both an optimal growth temperature ≥ 60°C and an optimal pH of ≤ 4.0. A majority of the extremely thermoacidophilic species studied to date  belong to the archaeal orders Sulfolobales and Thermoplasmatales. From what is currently known, it is interesting that the most heat-tolerant extreme thermoacidophiles are not the most acid-tolerant and vice versa. The most thermophilic of the extreme thermoacidophiles, crenarcheon Acidianus infernus, grows at temperatures up to 95°C (Topt of 85–90°C) but at pHs only as low as 1.0 (pHopt 2.0). In contrast, Picrophilus species of the euryarchaeal order Thermoplasmatales are the most acidophilic, growing at pHs as low as 0 (pHopt 0.7), but at temperatures up to only 65°C (Topt of 60°C). Insights into life in hot acid may be soon forthcoming, since genome sequences exist or are underway for many extreme thermoacidophiles. Furthermore, several new species in known genera of Sulfolobales (Acidianus, Metallosphaera) have been reported, as well as a new member of the Thermoplasmatales, Thermogymnomonas acidicola. We may have only scratched the surface with respect to extreme thermoacidophile diversity, because new environments not previously known to harbor these microorganisms have recently been identified. For example, mathematical modeling and 16S rRNA data suggest that conditions conducive to thermoacidophilic growth exist in deep-sea hydrothermal vents, a hypothesis supported by reports of the first euryarchaeon from the order Deep-sea Hydrothermal Vent Euryarchaeotic 2 (DHVE2), Aciduliprofundum boonei. Although A. boonei (Topt 70°C) grows best at a pH slightly above 4.0, 16S rRNA indicates that tHis microorganism comprises 10–15% of selected vent-associated archaeal populations, suggesting that there are extreme thermoacidophiles from these sites yet to be isolated.

Mechanisms of resistance to and survival in hot acid

The mechanisms by which microbial life thrives in hot acid have been investigated in some detail in recent years, triggered by the availability of genome sequence data, functional genomics tools, and molecular genetics. While the intrinsic basis for this novel growth physiology is not clear, clues are emerging as to how these microorganisms survive in the face of hot, acidic, and often metal-laden conditions which are typically associated with DNA damage, protein denaturation, and other disruptions in cellular processes.

DNA damage and repair

High temperatures and the potential for cytosol acidification heighten the possibility of DNA damage or modification in extreme thermoacidophiles relative to mesophilic neutrophiles. Thus, clues to DNA damage repair may emerge from examination of this cellular function in hot acid biotopes. It is surprising that basal mutation rates for extreme thermoacidophiles are not particularly high. For example, Sulfolobus acidocaldarius has a spontaneous mutation rate similar to that of E. coli. Furthermore, when Sulfolobus solfataricus and S. acidocaldarius were exposed to UV-irradiation, no significant increase in transcription of known DNA repair proteins was noted; however, it is possible that these genes are constitutively transcribed at higher levels than in mesophiles. Following irradiation, aggregates resembling those formed during plasmid-mediated conjugation were found, spurring speculation that Sulfolobus species may use conjugational DNA exchange and homologous recombination to repair mutated DNA. In a related study, S. solfataricus infected with the Sulfolobus spindle-shaped virus (SSV1) exhibited a similar, but heightened, response to UV-induced DNA damage, suggesting that viruses may be an evolutionary component of stress management systems. Another spindle-shaped virus, SSV2 from native host “Sulfolobus islandicus” REY15/4, sent infected “S. islandicus” REY15A cells into a metabolically inactive state upon encountering unfavorable environmental conditions and then played a role in re-starting metabolic activity once favorable growth conditions emerged. Up-regulation of two S. solfataricus recA/rad51 homologs (radA, SSO0250; radA-like, SSO0777) in response to a DNA-damaging antibiotic led to discovery of the first regulatory protein involved in archaeal DNA damage repair (Sta1, SSO0048) [21]. Neither the radA-like SSO0777, its sta1 activator, nor radA itself were induced by UV-irradiation, indicating that the nature of DNA damage may drive the specific type of repair response.

Heat shock

Though extreme thermoacidophiles thrive at temperatures up to 95°C, they are still susceptible to thermal stresses such that they exhibit both cold shock and heat shock responses. Extremely thermoacidophilic archaea react to supraoptimal temperatures in much the same way as other microorganisms. Most work to date has focused on the archaeal thermosome, or rosettasome, a heat-shock responsive HSP60-like molecular chaperone that has been implicated in many cellular roles. However, recent efforts have shown that heat shock response in extreme thermoacidophiles is extensive, involving much more than chaperones or other proteins involved in protein refolding. When exponentially growing S. solfataricus was shifted from its growth temperature optimum (80°C) to 90°C, approximately 1/3 of the transcriptome responded within 5 minutes. Included in this set of genes were many insertion elements and chromosomally encoded toxin-antitoxin (TA) loci - 22 TA pairs and 1 solitary toxin – all from the VapBC family. Chromosomally-encoded TA loci in bacteria are thought to be stress response elements, although the role of these tandem protein complexes in archaea has not been examined. Since PIN domain-containing VapCs, the “toxin” component of TA loci, are putative ribonucleases, these proteins could play an important role in post-transcriptional regulation in archaea, especially during heat shock.

Metal resistance

Extreme thermoacidophiles have developed mechanisms for tolerating heavy metals that are physiologically toxic to most microorganisms. These mechanisms involve their capacity to recover from metal-induced damage (similar to oxidative stress) and to limit the effective concentration of the toxic metal itself. In some cases, enzymes reduce or oxidize metals to less toxic forms - for example, the mercuric reductase in S. solfataricus reduces soluble intracellular Hg2+ to volatile elemental Hg0. In other cases, metal chelation or complexation can accomplish the same objective. In Sulfolobus metallicus, a polyphosphate (polyP)-based mechanism is believed to underlie cellular tolerance to high levels of copper; greater accumulation of polyP granules was observed in S. metallicus (considered to have higher levels of Cu tolerance) compared to S. solfataricus, and granule size was noted to decrease as Cu levels were increased. Other strategies, however, do not involve metal transformation, direct or indirect, and instead are based on exporting toxic metal ions via P-type ATPases. Evidence to date suggests that multiple systems can operate in parallel in extreme thermoacidophiles to provide cumulative tolerance, with particular strategies useful for multiple metals. For example, copper tolerance in Sulfolobus species involves efflux ATPases in addition to the polyP pathway, but the same ATPases also contribute to cadmium tolerance. There may also be some intrinsic redundancy in protecting against heavy metal toxicity. Disruption mutants lacking mercuric reductase and its regulator (merAR) were found to still exhibit some mer operon transcription. In fact, creation of a mutant with a disrupted regulator resulted in increased merA expression and consequently increased Hg2+ tolerance, underscoring the importance of understanding the regulation of resistance mechanisms with respect to engineering characteristics for bioprocesses. While traditional acclimation and/or spontaneous mutant generation approaches are still useful, direct genetic manipulation offers the possibility of conferring similar levels of metal tolerance increase in a systematic manner.