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

Identification of the ATPase Subunit of the Primary Maltose Transporter in the Hyperthermophilic Anaerobe \u3ci\u3eThermotoga maritima\u3c/i\u3e

Thermotoga maritima is a hyperthermophilic anaerobic bacterium that produces molecular hydrogen (H2) by fermentation. It catabolizes a broad range of carbohydrates through the action of diverse ABC transporters. However, in T. maritima and related species, highly similar genes with ambiguous annotation obscure a precise understanding of genome function. In T. maritima, three putative malK genes, all annotated as ATPase subunits, exhibited high identity to each other. To distinguish between these genes, malK disruption mutants were constructed by gene replacement, and the resulting mutant cell lines were characterized. Only a disruption of malK3 produced a defect in maltose catabolism. To verify that the mutant phenotype arose specifically from malK3 inactivation, the malK3 mutation was repaired by recombination, and maltose catabolism was restored. This study demonstrates the importance of a maltose ABC-type transporter and its relationship to sugar metabolism in T. maritima. IMPORTANCE: The application and further development of a genetic system was used here to investigate gene paralogs in the hyperthermophile Thermotoga maritima. The occurrence of three ABC transporter ATPase subunits all annotated as malK was evaluated using a combination of genetic and bioinformatic approaches. The results clarify the role of only one malK gene in maltose catabolism in a nonmodel organism noted for fermentative hydrogen production. Includes supplemental material

Carbohydrate Hydrolysis and Transport in the Extreme Thermoacidophile \u3ci\u3eSulfolobus solfataricus\u3c/i\u3e

Extremely thermoacidophilic microbes, such as Sulfolobus solfataricus, are strict chemoheterotrophs despite their geologic niche. To clarify their ecophysiology, the overlapping roles of endoglucanases and carbohydrate transporters were examined during growth on soluble cellodextrins as the sole carbon and energy source. Strain-specific differences in genome structure implied a unique role for one of three endogenous endoglucanases. Plasmid-based endoglucanase expression promoted the consumption of oligosaccharides, including cellohexaose (G6) through cellonanaose (G9). Protein transporters required for cellodextrin uptake were identified through mutagenesis and complementation of an ABC transporter cassette, including a putative oligosaccharide binding protein. In addition, ablation of the binding protein compromised growth on glucose and alpha-linked oligosaccharides while inactivation of a previously described glucose transporter had no apparent impact. These data demonstrate that S. solfataricus employs a redundant mechanism for soluble cellodextrin catabolism having both substrate uptake and extracytoplasmic hydrolytic components

Mercury Inactivates Transcription and the Generalized Transcription Factor TFB in the Archaeon \u3ci\u3eSulfolobus solfataricus\u3c/i\u3e

Mercury has a long history as an antimicrobial agent effective against eukaryotic and prokaryotic organisms. Despite its prolonged use, the basis for mercury toxicity in prokaryotes is not well understood. Archaea, like bacteria, are prokaryotes but they use a simplified version of the eukaryotic transcription apparatus. This study examined the mechanism of mercury toxicity to the archaeal prokaryote Sulfolobus solfataricus. In vivo challenge with mercuric chloride instantaneously blocked cell division, eliciting a cytostatic response at submicromolar concentrations and a cytocidal response at micromolar concentrations. The cytostatic response was accompanied by a 70% reduction in bulk RNA synthesis and elevated rates of degradation of several transcripts, including tfb-1, tfb-2, and lacS. Whole-cell extracts prepared from mercuric chloride-treated cells or from cell extracts treated in vitro failed to support in vitro transcription of 16S rRNAp and lacSp promoters. Extract-mixing experiments with treated and untreated extracts excluded the occurrence of negative-acting factors in the mercury-treated cell extracts. Addition of transcription factor B (TFB), a general transcription factor homolog of eukaryotic TFIIB, to mercury-treated cell extracts restored \u3e50% of in vitro transcription activity. Consistent with this finding, mercuric ion treatment of TFB in vitro inactivated its ability to restore the in vitro transcription activity of TFB-immunodepleted cell extracts. These findings indicate that the toxicity of mercuric ion in S. solfataricus is in part the consequence of transcription inhibition due to TFB-1 inactivation

A Comment on the Topological Phase for Anti-Particles in a Lorentz-violating environment

Recently, a scheme to analyse topological phases in Quantum Mechanics by means of the non-relativistic limit of fermions non-minimally coupled to a Lorentz-breaking background has been proposed. In this letter, we show that the fixed background, responsible for the Lorentz-symmetry violation, may induce opposite Aharonov-Casher phases for a particle and its corresponding antiparticle. We then argue that such a difference may be used to investigate the asymmetry for particle/anti-particle as well as to propose bounds on the associated Lorentz-symmetry violating parameters.Comment: 4 pages - A published versio

Targeted Disruption of the α-Amylase Gene in the Hyperthermophilic Archaeon \u3ci\u3eSulfolobus solfataricus\u3c/i\u3e

Sulfolobus solfataricus secretes an acid-resistant _-amylase (amyA) during growth on starch as the sole carbon and energy source. Synthesis of this activity is subject to catabolite repression. To better understand _-amylase function and regulation, the structural gene was identified and disrupted and the resulting mutant was characterized. Internal _-amylase peptide sequences obtained by tandem mass spectroscopy were used to identify the amyA coding sequence. Anti-_-amylase antibodies raised against the purified protein immunoprecipitated secreted _-amylase activity and verified the enzymatic identity of the sequenced protein. A new gene replacement method was used to disrupt the amyA coding sequence by insertion of a modified allele of the S. solfataricus lacS gene. PCR and DNA sequence analysis were used to characterize the altered amyA locus in the recombinant strain. The amyA::lacS mutant lost the ability to grow on starch, glycogen, or pullulan as sole carbon and energy sources. During growth on a non-catabolite-repressing carbon source with added starch, the mutant produced no detectable secreted amylase activity as determined by enzyme assay, plate assay, or Western blot analysis. These results clarify the biological role of the α-amylase and provide additional methods for the directed genetic manipulation of the S. solfataricus genome

Molecular Characterization of the α-Glucosidase Gene (\u3ci\u3emalA\u3c/i\u3e) from the Hyperthermophilic Archaeon \u3ci\u3eSulfolobus solfataricus\u3c/i\u3e

Acidic hot springs are colonized by a diversity of hyperthermophilic organisms requiring extremes of temperature and pH for growth. To clarify how carbohydrates are consumed in such locations, the structural gene (malA) encoding the major soluble α-glucosidase (maltase) and flanking sequences from Sulfolobus solfataricus were cloned and characterized. This is the first report of an α-glucosidase gene from the archaeal domain. malA is 2,083 bp and encodes a protein of 693 amino acids with a calculated mass of 80.5 kDa. It is flanked on the 5’ side by an unusual 1-kb intergenic region. Northern blot analysis of the malA region identified transcripts for malA and an upstream open reading frame located 5’ to the 1-kb intergenic region. The malA transcription start site was located by primer extension analysis to a guanine residue 8 bp 5’ of the malA start codon. Gel mobility shift analysis of the malA promoter region suggests that sequences 3’ to position 233, including a consensus archaeal TATA box, play an essential role in malA expression. malA homologs were detected by Southern blot analysis in other S. solfataricus strains and in Sulfolobus shibatae, while no homologs were evident in Sulfolobus acidocaldarius, lending further support to the proposed revision of the genus Sulfolobus. Phylogenetic analyses indicate that the closest S. solfataricus α-glucosidase homologs are of mammalian origin. Characterization of the recombinant enzyme purified from Escherichia coli revealed differences from the natural enzyme in thermostability and electrophoretic behavior. Glycogen is a substrate for the recombinant enzyme. Unlike maltose hydrolysis, glycogen hydrolysis is optimal at the intracellular pH of the organism. These results indicate a unique role for the S. solfataricus α-glucosidase in carbohydrate metabolism

Comparative kinetic modeling of growth and molecular hydrogen overproduction by engineered strains of \u3ci\u3eThermotoga maritima \u3c/i\u3e

Thermotoga maritima is an anaerobic hyperthermophilic bacterium known for its high amounts of hydrogen (H2) production. In the current study, the kinetic modeling was applied on the engineered strains of T. maritima that surpassed the natural H2 production limit. The study generated a kinetic model explaining H2 overproduction and predicted a continuous fermentation system. A Leudking-Piret equation-based model predicted that H2 production by Tma200 (0.217 mol-H2 g–1-biomass) and Tma100 (0.147 mol-H2 g–1-biomass) were higher than wild type (0.096 mol-H2 g–1 -biomass) with reduced rates of maltose utilization. Sensitivity analysis confirmed satisfactory fitting of the experimental data. The slow growth rates of Tma200 (0.550 h–1) and Tma100 (0.495 h–1) are compared with the wild type (0.663 h–1). A higher maintenance energy along with growth and non-growth H2 coefficients corroborate the higher H2 productivity of the engineered strains. The modeled data established a continuous fermentation system for the sustainable H2 production. (Inludes 2 supplemental figures

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

Regulation of Mercury Resistance in the Crenarchaeote \u3ci\u3eSulfolobus solfataricus\u3c/i\u3e

Mercuric ion, Hg(II), inactivates generalized transcription in the crenarchaeote Sulfolobus solfataricus. Metal challenge simultaneously derepresses transcription of mercuric reductase (merA) by interacting with the archaeal transcription factor aMerR. Northern blot and primer extension analyses identified two additional Hg(II)-inducible S. solfataricus genes, merH and merI (SSO2690), located on either side of merA. Transcription initiating upstream of merH at promoter merHp was metal inducible and extended through merA and merI, producing a merHAI transcript. Northern analysis of a merRA double mutant produced by linear DNA recombination demonstrated merHp promoter activity was dependent on aMerR to overcome Hg(II) transcriptional inhibition. Unexpectedly, in a merA disruption mutant, the merH transcript was transiently induced after an initial period of Hg(II)-mediated transcription inhibition, indicating continued Hg(II) detoxification. Metal challenge experiments using mutants created by markerless exchange verified the identity of the MerR binding site as an inverted repeat (IR) sequence overlapping the transcription factor B binding recognition element of merHp. The interaction of recombinant aMerR with merHp DNA, studied using electrophoretic mobility shift analysis, demonstrated that complex formation was template specific and dependent on the presence of the IR sequence but insensitive to Hg(II) addition and site-specific IR mutations that relieved in vivo merHp repression. Despite containing a motif resembling a distant ArsR homolog, these results indicate aMerR remains continuously DNA bound to protect and coordinate Hg(II)-responsive control over merHAI transcription. The new genetic methods developed in this work will promote experimental studies on S. solfataricus and other Crenarchaeota