Survival of the Fittest: Overcoming Oxidative Stress at the Extremes of Acid, Heat and Metal

The habitat of metal respiring acidothermophilic lithoautotrophs is perhaps the most oxidizing environment yet identified. Geothermal heat, sulfuric acid and transition metals contribute both individually and synergistically under aerobic conditions to create this niche. Sulfuric acid and metals originating from sulfidic ores catalyze oxidative reactions attacking microbial cell surfaces including lipids, proteins and glycosyl groups. Sulfuric acid ����� ��������� ������������ ������������ ������������� ��� ���� ���������� ��� ������ �������� carbon. Oxidative reactions leading to abstraction of electrons is further impacted by heat through an increase in the proportion of reactant molecules with sufficient energy to react. Collectively these factors and particularly those related to metals must be overcome by thermoacidophilic lithoautotrophs in order for them to survive and proliferate. The necessary mechanisms to achieve this goal are largely unknown however mechanistics insights have been gained through genomic studies. This review focuses on the specific role of metals in this extreme environment with an emphasis on resistance mechanisms in Archaea

The Genome Sequence of the Metal-Mobilizing, Extremely Thermoacidophilic Archaeon \u3ci\u3eMetallosphaera sedula\u3c/i\u3e Provides Insights into Bioleaching-Associated Metabolism

Despite their taxonomic description, not all members of the order Sulfolobales are capable of oxidizing reduced sulfur species, which, in addition to iron oxidation, is a desirable trait of biomining microorganisms. However, the complete genome sequence of the extremely thermoacidophilic archaeon Metallosphaera sedula DSM 5348 (2.2 Mb, _2,300 open reading frames [ORFs]) provides insights into biologically catalyzed metal sulfide oxidation. Comparative genomics was used to identify pathways and proteins involved (directly or indirectly) with bioleaching. As expected, the M. sedula genome contains genes related to autotrophic carbon fixation, metal tolerance, and adhesion. Also, terminal oxidase cluster organization indicates the presence of hybrid quinol-cytochrome oxidase complexes. Comparisons with the mesophilic biomining bacterium Acidithiobacillus ferrooxidans ATCC 23270 indicate that the M. sedula genome encodes at least one putative rusticyanin, involved in iron oxidation, and a putative tetrathionate hydrolase, implicated in sulfur oxidation. The fox gene cluster, involved in iron oxidation in the thermoacidophilic archaeon Sulfolobus metallicus, was also identified. These iron- and sulfur-oxidizing components are missing from genomes of nonleaching members of the Sulfolobales, such as Sulfolobus solfataricus P2 and Sulfolobus acidocaldarius DSM 639. Whole-genome transcriptional response analysis showed that 88 ORFs were up-regulated twofold or more in M. sedula upon addition of ferrous sulfate to yeast extract-based medium; these included genes for components of terminal oxidase clusters predicted to be involved with iron oxidation, as well as genes predicted to be involved with sulfur metabolism. Many hypothetical proteins were also differentially transcribed, indicating that aspects of the iron and sulfur metabolism of M. sedula remain to be identified and characterized

Use of a Robust Dehydrogenase from an Archael Hyperthermophile in Asymmetric Catalysis–Dynamic Reductive Kinetic Resolution Entry into (S)-Profens

Hyperthermophilic archaea are of great interest in evolutionary microbiology, owing to their ability to withstand high temperatures, and often extremes of pressure, pH and salinity. Enzymes from these organisms1 may offer particular opportunities for asymmetric synthesis, complementary to approaches with mesophilic enzymes,2 or those involving enzyme3 and pathway4 reengineering. However, perhaps due to a bias that hyperthermophilic enzymes have “narrow substrate specificities,”5 archaeal extremophiles remain a largely untapped resource in asymmetric synthesis.6 Herein, we disclose a remarkably general Dynamic Reductive Kinetic Resolution (DYRKR) entry into (S)-profens, including several important NSAIDs. The enzyme employed is alcohol dehydrogenase (ADH)-10, one of 13 annotated ADHs in the hyperthermophile Sulfolobus solfataricus. Protein phylogenetic analysis of this paralogous family indicates SsADH-10 is most closely related to homologues in distant taxa (Fig. 1). The highest identity between SsADH-10 and any other SsADHs is only 34%, suggesting that the SsADH family was established prior to the emergence of other archaeal lineages. Though not described as such, the SsADH-10 appears to be the only SsADH isozyme for which structural information is available in the pdb.

Metal Resistance and Lithoautotrophy in the Extreme Thermoacidophile \u3ci\u3eMetallosphaera sedula\u3c/i\u3e

Archaea such as Metallosphaera sedula are thermophilic lithoautotrophs that occupy unusually acidic and metal-rich environments. These traits are thought to underlie their industrial importance for bioleaching of base and precious metals. In this study, a genetic approach was taken to investigate the specific relationship between metal resistance and lithoautotrophy during biotransformation of the primary copper ore, chalcopyrite (CuFeS2). In this study, a genetic system was developed for M. sedula to investigate parameters that limit bioleaching of chalcopyrite. The functional role of the M. sedula copRTA operon was demonstrated by cross-species complementation of a copper-sensitive Sulfolobus solfataricus copR mutant. Inactivation of the gene encoding the M. sedula copper efflux protein, copA, using targeted recombination compromised metal resistance and eliminated chalcopyrite bioleaching. In contrast, a spontaneous M. sedula mutant (CuR1) with elevated metal resistance transformed chalcopyrite at an accelerated rate without affecting chemoheterotrophic growth. Proteomic analysis of CuR1 identified pleiotropic changes, including altered abundance of transport proteins having AAA-ATPase motifs. Addition of the insoluble carbonate mineral witherite (BaCO3) further stimulated chalcopyrite lithotrophy, indicating that carbon was a limiting factor. Since both mineral types were actively colonized, enhanced metal leaching may arise from the cooperative exchange of energy and carbon between surface-adhered populations. Genetic approaches provide a new means of improving the efficiency of metal bioleaching by enhancing the mechanistic understanding of thermophilic lithoautotrophy

Use of a Robust Dehydrogenase from an Archael Hyperthermophile in Asymmetric Catalysis–Dynamic Reductive Kinetic Resolution Entry into (S)-Profens

Hyperthermophilic archaea are of great interest in evolutionary microbiology, owing to their ability to withstand high temperatures, and often extremes of pressure, pH and salinity. Enzymes from these organisms1 may offer particular opportunities for asymmetric synthesis, complementary to approaches with mesophilic enzymes,2 or those involving enzyme3 and pathway4 reengineering. However, perhaps due to a bias that hyperthermophilic enzymes have “narrow substrate specificities,”5 archaeal extremophiles remain a largely untapped resource in asymmetric synthesis.6 Herein, we disclose a remarkably general Dynamic Reductive Kinetic Resolution (DYRKR) entry into (S)-profens, including several important NSAIDs. The enzyme employed is alcohol dehydrogenase (ADH)-10, one of 13 annotated ADHs in the hyperthermophile Sulfolobus solfataricus. Protein phylogenetic analysis of this paralogous family indicates SsADH-10 is most closely related to homologues in distant taxa (Fig. 1). The highest identity between SsADH-10 and any other SsADHs is only 34%, suggesting that the SsADH family was established prior to the emergence of other archaeal lineages. Though not described as such, the SsADH-10 appears to be the only SsADH isozyme for which structural information is available in the pdb.

Phenotypic responses to interspecies competition and commensalism in a naturally derived microbial co-culture

The fundamental question of whether different microbial species will co-exist or compete in a given environment depends on context, composition and environmental constraints. Model microbial systems can yield some general principles related to this question. In this study we employed a naturally occurring co-culture composed of heterotrophic bacteria, Halomonas sp. HL-48 and Marinobacter sp. HL- 58, to ask two fundamental scientific questions: 1) how do the phenotypes of two naturally co-existing species respond to partnership as compared to axenic growth? and 2) how do growth and molecular phenotypes of these species change with respect to competitive and commensal interactions? We hypothesized – and confirmed – that co-cultivation under glucose as the sole carbon source would result in competitive interactions. Similarly, when glucose was swapped with xylose, the interactions became commensal because Marinobacter HL-58 was supported by metabolites derived from Halomonas HL- 48. Each species responded to partnership by changing both its growth and molecular phenotype as assayed via batch growth kinetics and global transcriptomics. These phenotypic responses depended on nutrient availability and so the environment ultimately controlled how they responded to each other. This simplified model community revealed that microbial interactions are context-specific and different environmental conditions dictate how interspecies partnerships will unfold

Phenotypic responses to interspecies competition and commensalism in a naturally derived microbial co-culture

The fundamental question of whether different microbial species will co-exist or compete in a given environment depends on context, composition and environmental constraints. Model microbial systems can yield some general principles related to this question. In this study we employed a naturally occurring co-culture composed of heterotrophic bacteria, Halomonas sp. HL-48 and Marinobacter sp. HL- 58, to ask two fundamental scientific questions: 1) how do the phenotypes of two naturally co-existing species respond to partnership as compared to axenic growth? and 2) how do growth and molecular phenotypes of these species change with respect to competitive and commensal interactions? We hypothesized – and confirmed – that co-cultivation under glucose as the sole carbon source would result in competitive interactions. Similarly, when glucose was swapped with xylose, the interactions became commensal because Marinobacter HL-58 was supported by metabolites derived from Halomonas HL- 48. Each species responded to partnership by changing both its growth and molecular phenotype as assayed via batch growth kinetics and global transcriptomics. These phenotypic responses depended on nutrient availability and so the environment ultimately controlled how they responded to each other. This simplified model community revealed that microbial interactions are context-specific and different environmental conditions dictate how interspecies partnerships will unfold

Norspermidine is not a self-produced trigger for biofilm disassembly

SummaryFormation of Bacillus subtilis biofilms, consisting of cells encapsulated within an extracellular matrix of exopolysaccharide and protein, requires the polyamine spermidine. A recent study reported that (1) related polyamine norspermidine is synthesized by B. subtilis using the equivalent of the Vibrio cholerae biosynthetic pathway, (2) exogenous norspermidine at 25 μM prevents B. subtilis biofilm formation, (3) endogenous norspermidine is present in biofilms at 50–80 μM, and (4) norspermidine prevents biofilm formation by condensing biofilm exopolysaccharide. In contrast, we find that, at concentrations up to 200 μM, exogenous norspermidine promotes biofilm formation. We find that norspermidine is absent in wild-type B. subtilis biofilms at all stages, and higher concentrations of exogenous norspermidine eventually inhibit planktonic growth and biofilm formation in an exopolysaccharide-independent manner. Moreover, orthologs of the V. cholerae norspermidine biosynthetic pathway are absent from B. subtilis, confirming that norspermidine is not physiologically relevant to biofilm function in this species

Mechanism of Catabolite Repression In The Archaeon Sulfolobus solfataricus

Sulfolobus solfataricus is an archaeon that thrives in a high temperature and low pH environment. Many Sulfolobus species have been isolated from terrestrial geothermal hot springs. In such places, the environmental factors greatly affect the temperature, pH, and availability of nutrients. To survive in such an environment, organisms must develop efficient strategies to obtain nutrients. Carbon catabolite repression (CCR) is a strategy to ensure optimal carbon metabolism. In this study, we employed Sulfolobus solfataricus as a model system to gain an understanding of the mechanisms of CCR in archaea. We isolated an S. solfataricus CCR mutant through screening of a lactose negative phenotype. Studies on this and related mutants showed that expression of lacS (β-glycosidase) was reduced during growth in repressing media and by mutation of an unlinked locus called car . The goal of this study was to identify key gene(s) or component(s) that control CCR in S. solfataricus. Extensive phenotypic study of CCR mutants revealed additional carbon metabolic defects associated with various sugars. In addition I found that the car mutation also resulted in changes in the expression level of several genes and proteins involved in chromatin remodeling, such as deacetylase and S-adenosylmethionine synthetase (metK). Genomic DNA library (BAC) screening and genome sequencing showed no apparent differences between the wild type and the car mutant. Taken together, my data show that the car mutation is pleiotropic and extragenic. To gain more insight into this phenomenon, chromatin DNA transformation and reconstitution experiments were conducted. The results revealed that the change in the chromatin dynamics affected the car CCR phenotype and that the CCR regulatory mechanism in S. solfataricus operates at the level of chromatin modification

Survival of the Fittest: Overcoming Oxidative Stress at the Extremes of Acid, Heat and Metal

The habitat of metal respiring acidothermophilic lithoautotrophs is perhaps the most oxidizing environment yet identified. Geothermal heat, sulfuric acid and transition metals contribute both individually and synergistically under aerobic conditions to create this niche. Sulfuric acid and metals originating from sulfidic ores catalyze oxidative reactions attacking microbial cell surfaces including lipids, proteins and glycosyl groups. Sulfuric acid also promotes hydrocarbon dehydration contributing to the formation of black “burnt” carbon. Oxidative reactions leading to abstraction of electrons is further impacted by heat through an increase in the proportion of reactant molecules with sufficient energy to react. Collectively these factors and particularly those related to metals must be overcome by thermoacidophilic lithoautotrophs in order for them to survive and proliferate. The necessary mechanisms to achieve this goal are largely unknown however mechanistics insights have been gained through genomic studies. This review focuses on the specific role of metals in this extreme environment with an emphasis on resistance mechanisms in Archaea