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High magnetic field Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers provide extremely high mass resolution (resolving power of ~200,000 at 400 m/z) protein detection across a broad mass range, enabling analysis of fine structure of isotopic peak clusters that is missed in other types of mass spectrometers. The protocol detailed here describes preparation of cellular extracts for purification of DNA-binding proteins using multiple chromatographic chemistries via fast protein liquid chromatography (FPLC), and identification and quantitation of the protein isoforms and their post-translational modifications by liquid chromatography coupled to Fourier transform ion cyclotron resonance mass spectrometry (LC-FT-ICR-MS). This protocol benefits from selectively purifying proteins for identification and quantitation by high resolution FT-ICR, which has the resolution to definitively distinguish between acetylation and trimethylation post-translational modification (PTM) additions.

Adaptive laboratory evolution (ALE) was employed to isolate arsenate and copper cross-resistant strains, from the copper-resistant M. sedula CuR1. The evolved strains, M. sedula ARS50-1 and M. sedula ARS50-2, contained 12 and 13 additional mutations, respectively, relative to M. sedula CuR1. Bioleaching capacity of a defined consortium (consisting of a naturally occurring strain and a genetically engineered copper sensitive strain) was increased by introduction of M. sedula ARS50-2, with 5.31 and 26.29% more copper recovered from enargite at a pulp density (PD) of 1 and 3% (w/v), respectively. M. sedula ARS50-2 arose as the predominant species and modulated the proportions of the other two strains after it had been introduced. Collectively, the higher Cu2+ resistance trait of M. sedula ARS50-2 resulted in a modulated microbial community structure, and consolidating enargite bioleaching especially at elevated PD.

Extremely thermoacidophilic Crenarchaeota belonging to the order Sulfolobales flourish in hot acidic habitats that are strongly oxidizing. The pH extremes of these habitats, however, often exceed the acid tolerance of type species and strains. Here, adaptive laboratory evolution was used over a 3-year period to test whether such organisms harbor additional thermoacidophilic capacity. Three distinct cell lines derived from a single type species were subjected to high-temperature serial passage while culture acidity was gradually increased. A 178-fold increase in thermoacidophily was achieved after 29 increments of shifted culture pH resulting in growth at pH 0.8 and 80°C. These strains were named super-acid-resistant Crenarchaeota (SARC). Mathematical modeling using growth parameters predicted the limits of acid resistance, while genome resequencing and transcriptome resequencing were conducted for insight into mechanisms responsible for the evolved trait. Among the mutations that were detected, a set of eight nonsynonymous changes may explain the heritability of increased acid resistance despite an unexpected lack of transposition. Four multigene components of the SARC transcriptome implicated oxidative stress as a primary challenge accompanying growth at acid extremes. These components included accelerated membrane biogenesis, induction of the mer operon, and an increased capacity for the generation of energy and reductant.

Epigenetic phenomena have not yet been reported in archaea, which are presumed to use a classical genetic process of heritability. Here, analysis of independent lineages of Sulfolobus solfataricus evolved for enhanced fitness implicated a non-Mendelian basis for trait inheritance. The evolved strains, called super acid-resistant Crenarchaeota (SARC), acquired traits of extreme acid resistance and genome stability relative to their wild-type parental lines. Acid resistance was heritable because it was retained regardless of extensive passage without selection. Despite the hereditary pattern, in one strain, it was impossible for these SARC traits to result from mutation because its resequenced genome had no mutation. All strains also had conserved, heritable transcriptomes implicated in acid resistance. In addition, they had improved genome stability with absent or greatly decreased mutation and transposition relative to a passaged control. A mechanism that would confer these traits without DNA sequence alteration could involve posttranslationally modified archaeal chromatin proteins. To test this idea, homologous recombination with isogenic DNA was used to perturb native chromatin structure. Recombination at up-regulated loci from the heritable SARC transcriptome reduced acid resistance and gene expression in the majority of recombinants. In contrast, recombination at a control locus that was not part of the heritable transcriptome changed neither acid resistance nor gene expression. Variation in the amount of phenotypic and expression changes across individuals was consistent with Rad54-dependent chromatin remodeling that dictated crossover location and branch migration. These data support an epigenetic model implicating chromatin structure as a contributor to heritable traits.

When carbohydrates are fermented by the hyperthermophilic anaerobe Thermotoga maritima, molecular hydrogen (H2) is formed in strict proportion to substrate availability. Excretion of the organic acids acetate and lactate provide an additional sink for removal of excess reductant. However, mechanisms controlling energy management of these metabolic pathways are largely unexplored. To investigate this topic, transient gene inactivation was used to block lactate production as a strategy to produce spontaneous mutant cell lines that overproduced H2 through mutation of unpredicted genetic targets. Single-crossover homologous chromosomal recombination was used to disrupt lactate dehydrogenase (encoded by ldh) with a truncated ldh fused to a kanamycin resistance cassette expressed from a native P groESL promoter. Passage of the unstable recombinant resulted in loss of the genetic marker and recovery of evolved cell lines, including strain Tma200. Relative to the wild type, and considering the mass balance of fermentation substrate and products, Tma200 grew more slowly, produced H2 at levels above the physiologic limit, and simultaneously consumed less maltose while oxidizing it more efficiently. Whole-genome resequencing indicated that the ABC maltose transporter subunit, encoded by malK3, had undergone repeated mutation, and high-temperature anaerobic [14C]maltose transport assays demonstrated that the rate of maltose transport was reduced. Transfer of the malK3 mutation into a clean genetic background also conferred increased H2 production, confirming that the mutant allele was sufficient for increased H2 synthesis. These data indicate that a reduced rate of maltose uptake was accompanied by an increase in H2 production, changing fermentation efficiency and shifting energy management.IMPORTANCE Biorenewable energy sources are of growing interest to mitigate climate change, but like other commodities with nominal value, require innovation to maximize yields. Energetic considerations constrain production of many biofuels, such as molecular hydrogen (H2) because of the competing needs for cell mass synthesis and metabolite formation. Here we describe cell lines of the extremophile Thermotoga maritima that exceed the physiologic limits for H2 formation arising from genetic changes in fermentative metabolism. These cell lines were produced using a novel method called transient gene inactivation combined with adaptive laboratory evolution. Genome resequencing revealed unexpected changes in a maltose transport protein. Reduced rates of sugar uptake were accompanied by lower rates of growth and enhanced productivity of H2.

Thermophilic and lithoautotrophic archaea such as Metallosphaera sedula occupy acidic, metal-rich environments and are used in biomining processes. Biotechnological approaches could accelerate these processes and improve metal recovery by biomining organisms, but systems for genetic manipulation in these organisms are currently lacking. To gain a better understanding of the interplay between metal resistance, autotrophy, and lithotrophic metabolism, a genetic system was developed for M. sedula and used to evaluate parameters governing the efficiency of copper bioleaching. Additionally, adaptive laboratory evolution was used to select for naturally evolved M. sedula cell lines with desirable phenotypes for biomining, and these adapted cell lines were shown to have increased bioleaching capacity and efficiency. Genomic methods were used to analyze mutations that led to resistance in the experimentally evolved cell lines, while transcriptomics was used to examine changes in stress-inducible gene expression specific to the environmental conditions.

Sulfolobus solfataricus is a thermoacidophilic member of the archaea whose envelope consists of an ether-linked lipid monolayer surrounded by a protein S-layer. Protein translocation across this envelope must accommodate a steep proton gradient that is subject to temperature extremes. To better understand this process in vivo, studies were conducted on the S. solfataricus glycosyl hydrolyase family 57 α-Amylase (AmyA). Cell lines harboring site specific modifications of the amyA promoter and AmyA structural domains were created by gene replacement using markerless exchange and characterized by Western blot, enzyme assay and culture-based analysis. Fusion of amyA to the malAp promoter overcame amyAp-mediated regulatory responses to media composition including glucose and amino acid repression implicating action act at the level of transcription. Deletion of the AmyA Class II N-terminal signal peptide blocked protein secretion and intracellular protein accumulation. Deletion analysis of a conserved bipartite C-terminal motif consisting of a hydrophobic region followed by several charged residues indicated the charged residues played an essential role in membrane-association but not protein secretion. Mutants lacking the C-terminal bipartite motif exhibited reduced growth rates on starch as the sole carbon and energy source, therefore, association of AmyA with the membrane improves carbohydrate utilization. Widespread occurrence of this motif in other secreted proteins of S. solfataricus and of related Crenarchaeota suggests protein association with membranes is a general trait used by these organisms to influence external processes.

Taurine (2-aminoethanesulfonic acid) is an amino acid-like compound widely distributed in animals and an essential nutrient in some species. Targeted metabolomics of marine and fresh water microalgae combined with medium supplementation identified biosynthetic pathway intermediates and necessary catalytic activities. Genomic analysis was then used to predict the first taurine biosynthetic pathway in these organisms. MRM-based electrospray ionization (ESI) LC–MS/MS analysis demonstrated that taurine is synthesized using a carbon backbone from l -serine combined with sulfur derived from sulfate. Metabolite analysis showed a non-uniform pattern in levels of pathway intermediates that were both species and supplement-dependent. While increased culture salinity raised taurine levels modestly in marine alga, taurine levels were strongly induced in a fresh water species implicating taurine as an organic osmolyte. Conservation of the synthetic pathway in algae and metazoans together with a pattern of intermittent distribution in other lineages suggests that it arose early in eukaryotic evolution. Elevated levels of cell-associated taurine in algae could offer a new and biorenewable source of this unusual bioactive compound.

This study investigates and presents the effects of thermodynamically coupled nonisothermal reaction-diffusion processes on microalgae growth, substrate consumption and neutral lipid production in a pond or wastewater treatment plant. The non-stirred chemostat hypothesis and linear nonequilibrium thermodynamics theory are applied to formulate the model equations that account the bulk phase compositions and temperature, resistances to the heat and mass transfers, and cross effects due to the thermodynamic coupling of heat and mass flow in the presence of chemical reaction. Nondimensional forms of the model equations are numerically solved. Bulk phase concentrations and temperatures, external resistances to heat and substrate transfers, and thermodynamic coupling may generate substantial number of new parameters that control the evolution and stability in microalgal growth and lipid production that are important for biofuels. Instabilities due to perturbations in nutrient concentrations may lead to spatial structures where the wavenumber plays important role in reaction diffusion systems.

Thermotoga maritima is a hyperthermophilic anaerobic bacterium that produces molecular hydrogen (H 2 ) 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.

Thermotoga maritima ferments a broad range of sugars to form acetate, carbon dioxide, traces of lactate, and near theoretic yields of molecular hydrogen (H 2 ). In this organism, the catabolism of pentose sugars such as arabinose depends on the interaction of the pentose phosphate pathway with the Embden-Myerhoff and Entner-Doudoroff pathways. Although the values for H 2 yield have been determined using pentose-supplemented complex medium and predicted by metabolic pathway reconstruction, the actual effect of pathway elimination on hydrogen production has not been reported due to the lack of a genetic method for the creation of targeted mutations. Here, a spontaneous and genetically stable pyrE deletion mutant was isolated and used as a recipient to refine transformation methods for its repair by homologous recombination. To verify the occurrence of recombination and to assess the frequency of crossover events flanking the deleted region, a synthetic pyrE allele, encoding synonymous nucleotide substitutions, was used. Targeted inactivation of araA (encoding arabinose isomerase) in the pyrE mutant was accomplished using a divergent, codon-optimized Thermosipho africanus pyrE allele fused to the T. maritima groES promoter as a genetic marker. Mutants lacking araA were unable to catabolize arabinose in a defined medium. The araA mutation was then repaired using targeted recombination. Levels of synthesis of H 2 using arabinose-supplemented complex medium by wild-type and araA mutant cell lines were compared. The difference between strains provided a direct measurement of H 2 production that was dependent on arabinose consumption. Development of a targeted recombination system for genetic manipulation of T. maritima provides a new strategy to explore H 2 formation and life at an extremely high temperature in the bacterial domain. IMPORTANCE We describe here the development of a genetic system for manipulation of Thermotoga maritima . T. maritima is a hyperthermophilic anaerobic bacterium that is well known for its efficient synthesis of molecular hydrogen (H 2 ) from the fermentation of sugars. Despite considerable efforts to advance compatible genetic methods, chromosome manipulation has remained elusive and hindered use of T. maritima or its close relatives as model hyperthermophiles. Lack of a genetic method also prevented efforts to manipulate specific metabolic pathways to measure their contributions to H 2 yield. To overcome this barrier, a homologous chromosomal recombination method was developed and used to characterize the contribution of arabinose catabolism to H 2 formation. We report here a stable genetic method for a hyperthermophilic bacterium that will advance studies on the basic and synthetic biology of Thermotogales .

Thermoacidophilic archaea, such as Metallosphaera sedula, are lithoautotrophs that occupy metal-rich environments. In previous studies, an M. sedula mutant lacking the primary copper efflux transporter, CopA, became copper sensitive. In contrast, the basis for supranormal copper resistance remained unclear in the spontaneous M. sedula mutant, CuR1. Here, transcriptomic analysis of copper-shocked cultures indicated that CuR1 had a unique regulatory response to metal challenge corresponding to the upregulation of 55 genes. Genome resequencing identified 17 confirmed mutations unique to CuR1 that were likely to change gene function. Of these, 12 mapped to genes with annotated function associated with transcription, metabolism, or transport. These mutations included 7 nonsynonymous substitutions, 4 insertions, and 1 deletion. One of the insertion mutations mapped to pseudogene Msed_1517 and extended its reading frame an additional 209 amino acids. The extended mutant allele was identified as a homolog of Pho4, a family of phosphate symporters that includes the bacterial PitA proteins. Orthologs of this allele were apparent in related extremely thermoacidophilic species, suggesting M. sedula naturally lacked this gene. Phosphate transport studies combined with physiologic analysis demonstrated M. sedula PitA was a low-affinity, high-velocity secondary transporter implicated in copper resistance and arsenate sensitivity. Genetic analysis demonstrated that spontaneous arsenate-resistant mutants derived from CuR1 all underwent mutation in pitA and nonselectively became copper sensitive. Taken together, these results point to archaeal PitA as a key requirement for the increased metal resistance of strain CuR1 and its accelerated capacity for copper bioleaching.

Living systems must continuously receive substrates from subenvironment, and the population metabolic rate model is affected on this flow of substrates to be metabolized, its relevant variables, and the rate at which operates. This study focuses on the influences of resistances and bulk phase factors with in the subenvironment and by thermodynamic coupling on reaction-transport processes representing a simple enzymatic conversion of a substrate to a product. Thermodynamic coupling refers to mass flow, or a reaction velocity that occurs without or opposite to the direction imposed by its primary thermodynamic driving force. We considered the effects of (i) subenvironment resistances for the heat and mass flows of reacting substrate in the form of the ratios of Sherwood to Nusselt numbers, (ii) the subenvironment bulk phase temperatures and concentration of substrate, and (iii) the cross-coefficients responsible for the induced effects due to the thermodynamic coupling. In order to study these effects, the ...

Most archaea and bacteria use a modified C in the anticodon wobble position of isoleucine tRNA to base pair with A but not with G of the mRNA. This allows the tRNA to read the isoleucine codon AUA without also reading the methionine codon AUG. To understand why a modified C, and not U or modified U, is used to base pair with A, we mutated the C34 in the anticodon of Haloarcula marismortui isoleucine tRNA (tRNA2Ile) to U, expressed the mutant tRNA in Haloferax volcanii, and purified and analyzed the tRNA. Ribosome binding experiments show that although the wild-type tRNA2Ile binds exclusively to the isoleucine codon AUA, the mutant tRNA binds not only to AUA but also to AUU, another isoleucine codon, and to AUG, a methionine codon. The G34 to U mutant in the anticodon of another H. marismortui isoleucine tRNA species showed similar codon binding properties. Binding of the mutant tRNA to AUG could lead to misreading of the AUG codon and insertion of isoleucine in place of methionine. This result would explain why most archaea and bacteria do not normally use U or a modified U in the anticodon wobble position of isoleucine tRNA for reading the codon AUA. Biochemical and mass spectrometric analyses of the mutant tRNAs have led to the discovery of a new modified nucleoside, 5-cyanomethyl U in the anticodon wobble position of the mutant tRNAs. 5-Cyanomethyl U is present in total tRNAs from euryarchaea but not in crenarchaea, eubacteria, or eukaryotes., National Institutes of Health (U.S.) (GM17151), National Institutes of Health (U.S.) (GM22854), National Institutes of Health (U.S.) (ES017010), Singapore-MIT Alliance for Research and Technology, Singapore. National Research Foundation, United States. Dept. of Energy (DE-FG36-08GO88055)

Microbial community ecology is an area of rapid growth centered within the larger discipline of microbiology. Newly developed research methods using molecular strategies have transformed this area into an accessible research topic. Despite such advances, transmission of this topic into pedagogical form has lagged behind. To improve this situation, an undergraduate research team created an artificial microbial community for class room use. They used color-coded enterobacterial taxa transformed with broad-host range plasmids that encoded green fluorescent protein color variants. Using this instructional tool, a class room teaching module was developed about microbial fitness. Over a multi-semester period, the module was introduced into a conventional microbiology curriculum and refined. The learning outcomes for this module include; understanding community composition, that the members of a community can respond in different ways to external events and, that these responses can be used to measure fitness. Learning outcomes were measured through pre and post testing and indicated a gain in understanding about microbial communities.

Extremely thermoacidophilic members of the Archaea such as the lithoautotroph, Metallosphaera sedula, are among the most acid resistant forms of life and are of great relevance in bioleaching. Here, adaptive laboratory evolution was used to enhance the acid resistance of this organism while genomics and transcriptomics were used in an effort to understand the molecular basis for this trait. Unlike the parental strain, the evolved derivative, M. sedula SARC-M1, grew well at pH of 0.90. Enargite (Cu3AsS4) bioleaching conducted at pH 1.20 demonstrated SARC-M1 leached 23.78 % more copper relative to the parental strain. Genome re-sequencing identified two mutations in SARC-M1 including a nonsynonymous mutation in Msed_0408 (an amino acid permease) and a deletion in pseudogene Msed_1517. Transcriptomic studies by RNA-seq of wild type and evolved strains at various low pH values demonstrated there was enhanced expression of genes in M. sedula SARC-M1 encoding membrane complexes and enzymes that extrude protons or that catalyze proton-consuming reactions. In addition, M. sedula SARC-M1 exhibited reduced expression of genes encoding enzymes that catalyze proton-generating reactions. These unique genomic and transcriptomic features support a model for increased acid resistance arising from enhanced control over cytoplasmic pH.

Experimental microbial evolution (EME) and its variant, adaptive laboratory evolution (ALE), are emerging empirical strategies for understanding fundamental biological processes in microbes. Integration of high throughput analytical methods combined with genetic selection leverage the power of these methods, particularly ALE, for the production of new biological traits while providing insight into their mechanistic basis. Though traditionally applied to model organisms, in this chapter, these methods are extended to studies using microbial extremophiles with an emphasis on current studies from our laboratory because of the near absence of published literature. Theoretic considerations are presented first. These are followed by descriptions of technologies that are required to extend these evolutionary methods to the study of extremophiles. Finally, the application of ALE is demonstrated using two distinct types of extremophiles. These include the hyperthermophilic anaerobic bacterium Thermotoga maritima, and the extremely thermoacidophilic archaeon Sulfolobus solfataricus. For T. maritima, the evolutionary genomics of deletion formation is presented, and for S. solfataricus, the role of insertion sequence elements is considered during the evolution of increased thermoacidophily. These examples demonstrate the utility of experimental evolutionary methods in association with extremophiles.

Thermoacidophilic archaea are found in heavy metal-rich environments, and, in some cases, these microorganisms are causative agents of metal mobilization through cellular processes related to their bioenergetics. Given the nature of their habitats, these microorganisms must deal with the potentially toxic effect of heavy metals. Here, we show that two thermoacidophilic Metallosphaera species with nearly identical (99.99%) genomes differed significantly in their sensitivity and reactivity to uranium (U). Metallosphaera prunae , isolated from a smoldering heap on a uranium mine in Thüringen, Germany, could be viewed as a “spontaneous mutant” of Metallosphaera sedula , an isolate from Pisciarelli Solfatara near Naples. Metallosphaera prunae tolerated triuranium octaoxide (U 3 O 8 ) and soluble uranium [U(VI)] to a much greater extent than M. sedula . Within 15 min following exposure to “U(VI) shock,” M. sedula , and not M. prunae , exhibited transcriptomic features associated with severe stress response. Furthermore, within 15 min post-U(VI) shock, M. prunae , and not M. sedula , showed evidence of substantial degradation of cellular RNA, suggesting that transcriptional and translational processes were aborted as a dynamic mechanism for resisting U toxicity; by 60 min post-U(VI) shock, RNA integrity in M. prunae recovered, and known modes for heavy metal resistance were activated. In addition, M. sedula rapidly oxidized solid U 3 O 8 to soluble U(VI) for bioenergetic purposes, a chemolithoautotrophic feature not previously reported. M. prunae , however, did not solubilize solid U 3 O 8 to any significant extent, thereby not exacerbating U(VI) toxicity. These results point to uranium extremophily as an adaptive, rather than intrinsic, feature for Metallosphaera species, driven by environmental factors.

This study focuses on the cell growth and the neutral lipid production modeling of Chlamydomonas reinhardtii in terms of different ammonium concentrations. Autotrophy was maintained during growth in a double walled bioreactor, using Tris Phosphate (TP medium) with only CO(2) and NH(4)Cl as sole sources of carbon and nitrogen, respectively. Nitrogen depletion results in an increase in neutral lipid production with an indirect effect on the growth of algal cells. Modified Baranyi-Roberts and logistic equations were used to describe the cell growth whereas Luedeking-Piret equation was used for neutral lipid production kinetics. Sensitivity analysis shows that the model equations satisfactorily predict the cell growth and lipid production. Based on the mathematical model predictions, growing algal cells in higher ammonium containing medium initially and switching to low ammonium containing medium in a later stage may result in elevated amounts of lipid production, which may be used for scale up and commercialization.

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.

Mercury is a heavy metal and toxic to all forms of life. Metal exposure can invoke a response to improve survival. In archaea, several components of a mercury response system have been identified, but it is not known whether metal transport is a member of this system. To identify such missing components, a peptide-tagged MerR transcription factor was used to localize enriched chromosome regions by chromosome immunoprecipitation combined with DNA sequence analysis. Such regions could serve as secondary regulatory binding sites to control the expression of additional genes associated with mercury detoxification. Among the 31 highly enriched loci, a subset of five was pursued as potential candidates based on their current annotations. Quantitative reverse transcription-PCR analysis of these regions with and without mercury treatment in WT and mutant strains lacking merR indicated significant regulatory responses under these conditions. Of these, a Family 5 extracellular solute-binding protein and the MarR transcription factor shown previously to control responses to oxidation were most strongly affected. Inactivation of the solute-binding protein by gene disruption increased the resistance of mutant cells to mercury challenge. Inductively coupled plasma-MS analysis of the mutant cell line following metal challenge indicated there was less intracellular mercury compared with the isogenic WT strain. Together, these regulated genes comprise new members of the archaeal MerR regulon and reveal a cascade of transcriptional control not previously demonstrated in this model organism.

Domestic ethanol production in the USA relies on starch feedstocks using a first generation bioprocess. Enzymes that contribute to this industry remain of critical value in new and established markets as commodity additives and for in planta production. A transition to non-food feedstocks is both desirable and essential to enable larger scale production. This objective would relieve dependence on foreign oil and strengthen the national economy. Feedstocks derived from corn stover, wheat straw, perennial grasses and timber require pretreatment to increase the accessibility of the cellulosic and hemicellulosic substrates to commodity enzymes for saccharification, which is followed by fermentation-based conversion of monosaccharides to ethanol. Hot acid pretreatment is the industrial standard method used to achieve deconstruction of lignocellulosic biomass. Therefore, enzymes that tolerate both acid and heat may contribute toward the improvement of lignocellulosic biomass processing. These enzymes are produced naturally by extremely thermophilic microbes, sometimes called extremophiles. This review summarizes information on enzymes from selected (acido)thermophiles that mediate saccharification of alpha- and beta-linked carbohydrates of relevance to biomass processing.

Modification of the cytidine in the first anticodon position of the AUA decoding tRNA Ile ( ) of bacteria and archaea is essential for this tRNA to read the isoleucine codon AUA and to differentiate between AUA and the methionine codon AUG. To identify the modified cytidine in archaea, we have purified this tRNA species from Haloarcula marismortui , established its codon reading properties, used liquid chromatography–mass spectrometry (LC-MS) to map RNase A and T1 digestion products onto the tRNA, and used LC-MS/MS to sequence the oligonucleotides in RNase A digests. These analyses revealed that the modification of cytidine in the anticodon of adds 112 mass units to its molecular mass and makes the glycosidic bond unusually labile during mass spectral analyses. Accurate mass LC-MS and LC-MS/MS analysis of total nucleoside digests of the demonstrated the absence in the modified cytidine of the C2-oxo group and its replacement by agmatine (decarboxy-arginine) through a secondary amine linkage. We propose the name agmatidine, abbreviation C + , for this modified cytidine. Agmatidine is also present in Methanococcus maripaludis and in Sulfolobus solfataricus total tRNA, indicating its probable occurrence in the AUA decoding tRNA Ile of euryarchaea and crenarchaea. The identification of agmatidine shows that bacteria and archaea have developed very similar strategies for reading the isoleucine codon AUA while discriminating against the methionine codon AUG.

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.

Flagellation in archaea is widespread and is involved in swimming motility. Here, we demonstrate that the structural flagellin gene from the crenarchaeaon Sulfolobus solfataricus is highly expressed in stationary-phase-grown cells and under unfavorable nutritional conditions. A mutant in a flagellar auxiliary gene, flaJ , was found to be nonmotile. Electron microscopic imaging of the flagellum indicates that the filaments are composed of right-handed helices.

Many biological toxins are known to attack specific cell types, delivering their enzymatic payloads to the cytosol. This process can be manipulated by molecular engineering of chimeric toxins. Using toxins with naturally unlinked components as a starting point is advantageous because it allows for the development of payloads separately from the binding/translocation components. Here the Clostridium botulinum C2 binding/translocation domain was retargeted to neural cell populations by deleting its non-specific binding domain and replacing it with a C. botulinum neurotoxin binding domain. This fusion protein was used to deliver fluorescently labeled payloads to Neuro-2a cells. Intracellular delivery was quantified by flow cytometry and found to be dependent on artificial enrichment of cells with the polysialoganglioside receptor GT1b. Visualization by confocal microscopy showed a dissociation of payloads from the early endosome indicating translocation of the chimeric toxin. The natural Clostridium botulinum C2 toxin was then delivered to human glioblastoma A172 and synchronized HeLa cells. In the presence of the fusion protein, native cytosolic enzymatic activity of the enzyme was observed and found to be GT1b-dependent. This retargeted toxin may enable delivery of therapeutics to peripheral neurons and be of use in addressing experimental questions about neural physiology.

Metallosphaera sedula is a thermoacidophilic crenarchaeote with a 2.19-Mb genome. Here, we report the genome sequences of several evolved derivatives of M. sedula generated through adaptive laboratory evolution for enhanced arsenate resistance.

Sulfolobus solfataricus is a thermoacidophilic crenarcheote with a 3.0-Mb genome. Here, we report the genome sequence of S. solfataricus strain 98/2, along with several evolved derivatives generated through experimental microbial evolution for enhanced thermoacidophily.

Thermotoga maritima is a hyperthermophilic bacterium with a small genome (1.86 Mbp). Genome resequencing of Tma200, a derivative produced by experimental microbial evolution, revealed the occurrence of deletions and substitution mutations. Their identification contributes to a better understanding of genome instability in this organism.

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 .

The production and consumption of molecular hydrogen drives the physiology and bioenergetics of many microorganisms in hydrothermal environments. As such, the potential of these microorganisms as model systems to probe fundamental issues related to biohydrogen production merits consideration. It is important to understand how carbon/energy sources relate to the disposition of reducing power and, ultimately, the formation of molecular hydrogen by high temperature microorganisms. This project focused on bacteria in the thermophilic order Thermotogales, fermentative anaerobes that produce H2 from simple and complex carbohydrates. The major thrusts of the project are summarized in the Objectives listed below: OBJECTIVE 1: Examine the regulation of substrate catabolic proteins and pathways as this relates to carbon partitioning, disposition of reducing power, and H2 generation in Thermotoga maritima. OBJECTIVE 2: Apply classical genetics and develop molecular genetic tools for Thermotoga species to dissect catabolic and regulatory pathways related to sugar metabolism and H2 evolution. OBJECTIVE 3: Thermotogales biodiversity arises from adaptive specialization that expands on a conserved minimal genome; physiological characterization of selected novel traits will be done to expand understanding of biohydrogenesis. Four species within the genus Thermotoga were examined to understand similarities and differences in the mechanisms by which simple and complexmore » carbohydrates were utilized and converted to molecular hydrogen. Although the core genome of these four species represented 75% of open reading frames (ORFs), there were significant differences in carbohydrate utilization patterns. New ABC transporters were identified within the Thermotogales through genomic and biochemical analysis. Molecular genetics tools were developed to examine Thermotoga maritima physiology. Cell lines were created in which both H2 and acetate levels were elevated on a per cell basis relative to the wild type, while lactate remained undetectable. Genome resequencing indicated that the primary genetic target for these phenotypic changes was the ATP binding component of a maltose ABC transporter. High temperature anaerobic [14C]-maltose transport assays demonstrated maltose uptake was reduced in the H2 overproducing cell lines. This suggested normal rates of maltose transport in the wild type organism lead to a metabolic imbalance that limited H2 synthesis. The microbial ecology of T. maritima was examined through functional genomics experiments. Under low nutrient conditions, T. maritima was observed to produce a range of putative peptides, some of which were related to α-carbon cyclic peptides produced by Bacillus subtilus. Finally, the role of ‘toga’ in these novel microorganisms was shown to involve association with insoluble growth substrates. The ‘toga’ distends from the cytoplasmic membrane-enclosed portion of the cells as they enter the late exponential/stationary phase of growth. Some of the genes encoding toga-associated proteins were up-regulated during this phase of growth and the distension is caused by continued growth of the toga, and not shrinkage of the cytoplasmic aspect of the cells. This increase in cell surface area may have selective value to provide a larger anchor for polysaccharide hydrolytic enzymes during a time of nutritional stress. This project led to many interesting insights about the Thermotogales that have both scientific and technological implications. Ongoing work will leverage these developments to further elucidate many interesting features of these novel microorganisms.« less

Mercury is a redox-active heavy metal that reacts with active thiols and depletes cellular antioxidants. Active resistance to the mercuric ion is a widely distributed trait among bacteria and results from the action of mercuric reductase (MerA). Protein phylogenetic analysis of MerA in bacteria indicated the occurrence of a second distinctive form of MerA among the archaea, which lacked an N-terminal metal recruitment domain and a C-terminal active tyrosine. To assess the distribution of the forms of MerA in an interacting community comprising members of both prokaryotic domains, studies were conducted at a naturally occurring mercury-rich geothermal environment. Geochemical analyses of Coso Hot Springs indicated that mercury ore (cinnabar) was present at concentrations of parts per thousand. Under high-temperature and acid conditions, cinnabar may be oxidized to the toxic form Hg 2+ , necessitating mercury resistance in resident prokaryotes. Culture-independent analysis combined with culture-based methods indicated the presence of thermophilic crenarchaeal and gram-positive bacterial taxa. Fluorescence in situ hybridization analysis provided quantitative data for community composition. DNA sequence analysis of archaeal and bacterial merA sequences derived from cultured pool isolates and from community DNA supported the hypothesis that both forms of MerA were present. Competition experiments were performed to assess the role of archaeal merA in biological fitness. An essential role for this protein was evident during growth in a mercury-contaminated environment. Despite environmental selection for mercury resistance and the proximity of community members, MerA retains the two distinct prokaryotic forms and avoids genetic homogenization.

A thermo-acidophilic Gram-positive bacterium, strain CsHg2T, which grows aerobically at 35–65 °C (optimum 55 °C) and at pH 2·0–6·0 (optimum 4·0), was isolated from a geothermal pool located in Coso Hot Springs in the Mojave Desert, California, USA. Phylogenetic analysis of 16S rRNA gene sequences showed that this bacterium was most closely related to the type strains ofAlicyclobacillus acidocaldarius(97·8 % identity) andAlicyclobacillus sendaiensis(96·9 %), three Japanese strains denoted as UZ-1, KHA-31 and MIH 332 (96·1–96·5 %) andAlicyclobacillusgenomic species FR-6 (96·3 %). Phenotypic characteristics including temperature and pH optima, G+C composition, acid production from a variety of carbon sources and sensitivity to different metal salts distinguished CsHg2TfromA. acidocaldarius,A. sendaiensisand FR-6. The cell lipid membrane was composed mainly ofω-cyclohexyl fatty acid, consistent with membranes from otherAlicyclobacillusspecies. Very low DNA–DNA hybridization values between CsHg2Tand the type strains ofAlicyclobacillusindicate that CsHg2Trepresents a distinct species. On the basis of these results, the nameAlicyclobacillus vulcanalissp. nov. is proposed for this organism. The type strain is CsHg2T(ATCC BAA-915T=DSM 16176T).

A culture-independent method called mRNA profiling has been developed for the analysis of fecal enterobacteria and their physiological status in environmental samples. This taxon-specific approach determines the single-cell content of selected gene transcripts whose abundance is either directly or inversely proportional to growth state. Fluorescence in situ hybridization using fluorochrome-labeled oligonucleotide probes was used to measure the cellular concentration of fis and dps mRNA. Relative levels of these transcripts provided a measure of cell growth state and the ability to enumerate fecal enterobacterial cell number. Orthologs were cloned by inverse PCR from several major enterobacterial genera, and probes specific for fecal enterobacteria were designed using multiple DNA sequence alignments. Probe specificity was determined experimentally using pure and mixed cultures of the major enterobacterial genera as well as secondary treated wastewater samples seeded with pure culture inocula. Analysis of the fecal enterobacterial community resident in unseeded secondary treated wastewater detected fluctuations in transcript abundance that were commensurate with incubation time and nutrient availability and demonstrated the utility of the method using environmental samples. mRNA profiling provides a new strategy to improve wastewater disinfection efficiency by accelerating water quality analysis.

An electric water heater was modified for large-scale cultivation of aerobic acidophilic hyperthermophiles to enable recovery of secreted proteins. Critical changes included thermostat replacement, redesign of the temperature control circuit, and removal of the cathodic anticorrosion system. These alterations provided accurate temperature and pH control. The bioreactor was used to cultivate selected strains of the archaeon Sulfolobus solfataricus and other species within this genus. Reformulation of a basal salts medium facilitated preparation of large culture volumes and eliminated sterilization-induced precipitation of medium components. Substrate induction of synthesis of the S . solfataricus -secreted alpha-amylase during growth in a defined medium supported the utility of the bioreactor for studies of physiologically regulated processes. An improved purification strategy was developed by using strong cation-exchange chromatography for recovery of the alpha-amylase and the processing of large sample volumes of acidic culture supernatant. These findings should simplify efforts to study acidophilic hyperthermophilic microbes and their secreted proteins.

The relative effects of three precise nitrogen limitation regimes on green micro-algae were assessed using the Trebouxiophycean alga Coccomyxa subellipsoidea grown in a chemostatic bioreactor system. The data provides further evidence that growth and triglyceride (TAG) accumulation are concurrent and independently proportional to the degree of nitrogen limitation in algae. Additionally, TAG accumulation was observed to proceed via oscillations with respect to time and percent dry weight quantity. The predator–prey model was applied to fit the experimental data and to obtain the physiological significance of these oscillations. The results determine the conditions of maximum neutral lipid productivity with respect to nitrate stress and highlight an area of potential future research.

In recent years several laboratories have developed effective plating techniques, identifying genetic markers that do not target cell wall synthesis, fusing archaeal promoters with recombinant genes, and isolating native vectors and promiscuous nonnative vectors. This chapter focuses on tractable systems that are currently available for the Archaea. Due to fundamental differences between gene transfer systems for each archaeal branch, the chapter is divided into three inclusive sections covering the halophilic and methanogenic Euryarchaeota and the hyperthermophilic Crenarchaeota. Despite varying degrees of difficulty growing Archaea, all three systems are routinely used by laboratories conducting research on archaeal genetics and can be mastered by anyone with a fundamental knowledge of microbial genetic techniques. Under low oxygen tension, Halobacterium sp. NRC-1 induces purple membrane patches in the cell membrane and buoyant gas vesicles intracellularly, which increases the availability of light and oxygen and allows a period of light-driven proton pumping and phototrophic growth. Targeted manipulation of the chromosome by directed recombination was recently added to the growing list of approaches for the genetic analysis of Sulfolobus solfataricus. Plasmids that do not replicate in S. solfataricus can be used to introduce DNA into the genome.

Few antibiotics targeting members of the archaeal domain are currently available for genetic studies. Since bacterial antibiotics are frequently directed against competing and related organisms, archaea by analogy might produce effective antiarchaeal antibiotics. Peptide antibiotic (halocin) preparations from euryarchaeal halophilic strains S8a, GN101, and TuA4 were found to be toxic for members of the hyperthermophilic crenarchaeal genus Sulfolobus . No toxicity was evident against representative bacteria or eukarya. Halocin S8 (strain S8a) and halocin R1 (strain GN101) preparations were cytostatic, while halocin A4 (strain TuA4) preparations were cytocidal. Subsequent studies focused on the use of halocin A4 preparations and Sulfolobus solfataricus . Strain TuA4 cell lysates were not toxic for S. solfataricus , and protease (but not nuclease) treatment of the halocin A4 preparation inactivated toxicity, indicating that the A4 toxic factor must be a secreted protein. Potassium chloride supplementation of the Sulfolobus assay medium potentiated toxicity, implicating use of a salt-dependent mechanism. The utility of halocin A4 preparations for genetic manipulation of S. solfataricus was assessed through the isolation of UV-induced resistant mutants. The mutants exhibited stable phenotypes and were placed into distinct classes based on their levels of resistance.

Commercial bioprocessing of plant carbohydrates, such as starch or cellulose, necessitates the use of commodity enzyme additives to accelerate polysaccharide hydrolysis. To simplify this procedure, transgenic plant tissues constitutively producing commodity enzymes were examined as a strategy for accelerating carbohydrate bioprocessing. Hyperthermophilic glycosyl hydrolases were selected to circumvent enzyme toxicity, because such enzymes are inactive at plant growth temperatures and are therefore physiologically benign. Transgenic tobacco lines were established that produced either a hyperthermophilic α-glucosidase or a β-glycosidase using genes derived from the archaeon Sulfolobus solfataricus. Western blot and immunoprecipitation analyses were used to demonstrate the presence of recombinant enzymes in plant tissues. Transgenic enzyme levels exhibited an unusual delayed pattern of accumulation while their activities survived plant tissue preservation. Transgenic plant protein extracts released glucose from purified polysaccharide substrates at appreciable rates during incubation in high-temperature reactions. Glucose was also produced following enzymatic treatment of plant extracts enriched for endogenous polysaccharides. Direct conversion of plant tissue into free sugar was evident using whole plant extracts of either transgenic line, and could be significantly accelerated in a synergistic manner by combining transgenic line extracts. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 70: 151–159, 2000.

Municipal effluent is the largest reservoir of human enteric bacteria. Its public health significance, however, depends upon the physiological status of the wastewater bacterial community. A novel immunofluorescence assay was developed and used to examine the bacterial growth state during wastewater disinfection. Quantitative levels of three highly conserved cytosolic proteins (DnaK, Dps, and Fis) were determined by using enterobacterium-specific antibody fluorochrome-coupled probes. Enterobacterial Fis homologs were abundant in growing cells and nearly undetectable in stationary-phase cells. In contrast, enterobacterial Dps homologs were abundant in stationary-phase cells but virtually undetectable in growing cells. The range of variation in the abundance of both proteins was at least 100-fold as determined by Western blotting and immunofluorescence analysis. Enterobacterial DnaK homologs were nearly invariant with growth state, enabling their use as permeabilization controls. The cellular growth states of individual enterobacteria in wastewater samples were determined by measurement of Fis, Dps, and DnaK abundance (protein profiling). Intermediate levels of Fis and Dps were evident and occurred in response to physiological transitions. The results indicate that chlorination failed to kill coliforms but rather elicited nutrient starvation and a reversible nonculturable state. These studies suggest that the current standard procedures for wastewater analysis which rely on detection of culturable cells likely underestimate fecal coliform content.

The hyperthermophilic archaeon Sulfolobus solfataricus employs a catabolite repression-like regulatory system to control enzymes involved in carbon and energy metabolism. To better understand the basis of this system, spontaneous glycosyl hydrolase mutants were isolated using a genetic screen for mutations, which reduced expression of the lacS gene. The specific activities of three glycosyl hydrolases, including an α-glucosidase (malA), a β-glycosidase (lacS), and the major secreted α-amylase, were measured in the mutant strains using enzyme activity assays, Western blot analysis, and Northern blot analysis. On the basis of these results the mutants were divided into two classes. Group I mutants exhibited a pleiotropic defect in glycosyl hydrolase expression, while a single group II mutant was altered only in lacS expression. PCR, Southern blot analysis, comparative heterologous expression in Escherichia coli, and DNA sequence analysis excluded cis-acting mutations as the explanation for reduced lacS expression in group I mutants. In contrast lacS and flanking sequences were deleted in the group II mutant. Revertants were isolated from group I mutants using a lacS-specific screen and selection. These revertants were pleiotropic and restored glycosyl hydrolase activity either partially or completely to wild-type levels as indicated by enzyme assays and Western blots. The lacS mutation in the group II mutant, however, was nonrevertible. The existence of group I mutants and their revertants reveals the presence of a trans-acting transcriptional regulatory system for glycosyl hydrolase expression.

We have identified maize (Zea mays L. inbred B73) mitochondrial homologs of the Escherichia coli molecular chaperones DnaK (HSP70) and GroEL (cpn60) using two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblots. During heat stress (42°C for 4 h), levels of HSP70 and cpn60 proteins did not change significantly. In contrast, levels of two 22-kD proteins increased dramatically (HSP22). Monoclonal antibodies were developed to maize HSP70, cpn60, and HSP22. The monoclonal antibodies were characterized with regard to their cross-reactivity to chloroplastic, cytosolic, and mitochondrial fractions, and to different plant species. Expression of mitochondrial HSP22 was evaluated with regard to induction temperature, time required for induction, and time required for degradation upon relief of stress. Maximal HSP22 expression occurred in etiolated seedling mitochondria after 5 h of a +13°C heat stress. Upon relief of heat stress, the HSP22 proteins disappeared with a half-life of about 4 h and were undetectable after 21 h of recovery. Under continuous heat-stress conditions, the level of HSP22 remained high. A cDNA for maize mitochondrial HSP22 was cloned and extended to full length with sequences from an expressed sequence tag database. Sequence analysis indicated that HSP22 is a member of the plant small heat-shock protein superfamily.

DnaK is essential for starvation-induced resistance to heat, oxidation, and reductive division in Escherichia coli . Studies reported here indicate that DnaK is also required for starvation-induced osmotolerance, catalase activity, and the production of the RpoS-controlled Dps (PexB) protein. Because these dnaK mutant phenotypes closely resemble those of rpoS (ς 38 ) mutants, the relationship between DnaK and RpoS was evaluated directly during growth and starvation at 30°C in strains with genetically altered DnaK content. A starvation-specific effect of DnaK on RpoS abundance was observed. During carbon starvation, DnaK deficiency reduced RpoS levels threefold, while DnaK excess increased RpoS levels nearly twofold. Complementation of the dnaK mutation restored starvation-induced RpoS levels to normal. RpoS deficiency had no effect on the cellular concentration of DnaK, revealing an epistatic relationship between DnaK and RpoS. Protein half-life studies conducted at the onset of starvation indicate that DnaK deficiency significantly destabilized RpoS. RpoH (ς 32 ) suppressors of the dnaK mutant with restored levels of RpoS and dnaK rpoS double mutants were used to show that DnaK plays both an independent and an RpoS-dependent role in starvation-induced thermotolerance. The results suggest that DnaK coordinates sigma factor levels in glucose-starved E. coli.

Crenarchaeota include extremely thermoacidophilic organisms that thrive in geothermal environments dominated by sulfidic ores and heavy metals such as mercury. Mercuric ion, Hg(II), inactivates transcription in the crenarchaeote Sulfolobus solfataricus and simultaneously derepresses transcription of a resistance operon, merHAI, through interaction with the MerR transcription factor. While mercuric reductase (MerA) is required for metal resistance, the role of MerH, an adjacent small and predicted product of an ORF, has not been explored. Inactivation of MerH either by nonsense mutation or by in-frame deletion diminished Hg(II) resistance of mutant cells. Promoter mapping studies indicated that Hg(II) sensitivity of the merH nonsense mutant arose through transcriptional polarity, and its metal resistance was restored partially by single copy merH complementation. Since MerH was not required in vitro for MerA-catalysed Hg(II) reduction, MerH may play an alternative role in metal resistance. Inductively coupled plasma-mass spectrometry analysis of the MerH deletion strain following metal challenge indicated that there was prolonged retention of intracellular Hg(II). Finally, a reduced rate of mer operon induction in the merH deletion mutant suggested that the requirement for MerH could result from metal trafficking to the MerR transcription factor.

An alpha-amylase was purified from culture supernatants of Sulfolobus solfataricus 98/2 during growth on starch as the sole carbon and energy source. The enzyme is a homodimer with a subunit mass of 120 kDa. It catalyzes the hydrolysis of starch, dextrin, and alpha-cyclodextrin with similar efficiencies. Addition of exogenous glucose represses production of alpha-amylase, demonstrating that a classical glucose effect is operative in this organism. Synthesis of [35S]-alpha-amylase protein is also subject to the glucose effect. alpha-Amylase is constitutively produced at low levels but can be induced further by starch addition. The absolute levels of alpha-amylase detected in culture supernatants varied greatly with the type of sole carbon source used to support growth. Aspartate was identified as the most repressing sole carbon source for alpha-amylase production, while glutamate was the most derepressing. The pattern of regulation of alpha-amylase production seen in this organism indicates that a catabolite repression-like system is present in a member of the archaea.

Overproduction of DnaK in Escherichia coli results in a bacteriocidal effect. This effect is most acute in stationary phase cells. A selection scheme was developed to isolate multicopy suppressors from an E. coli plasmid expression library, which overcome the stationary phase toxicity of excess DnaK. Two suppressor plasmids were recovered which contained inserts of 1.85 kb and 2.69 kb, respectively. Rearranged and deleted plasmid derivatives were constructed and used to further localize the suppressors. DNA sequence analysis demonstrated that one suppressor encoded phosphogluconate dehydratase (Edd) while the other suppressor encoded the N-terminal 237 amino acids of DnaK itself (DnaK'). Strains bearing the suppressor plasmids constitutively overproduced proteins with apparent masses of 66 kDa (Edd) and 37 kDa (DnaK') as determined by gel electrophoresis. Western blot analysis using polyclonal antisera specific for either Edd or DnaK confirmed the identity of these overproduced proteins. Suppression of DnaK toxicity was eliminated by the introduction of a + 1 frameshift mutation early in the respective coding regions of either of the two suppressors. These results suggest that suppressor gene translation plays a role in the mechanism of DnaK suppression.

During a 3-day period, glucose starvation of wild-type Escherichia coli produced thermotolerant, H2O2-resistant, small cells with a round morphology. These cells contained elevated levels of the DnaK protein, adjusted either for total protein or on a per-cell basis. Immunoprecipitation of [35S]methionine-labeled protein produced by such starving cells demonstrated that DnaK underwent continuous synthesis but at decreasing rates throughout this time. Glucose resupplementation of starving cells resulted in rapid loss of thermotolerance, H2O2 resistance, and the elevated DnaK levels. A dnaK deletion mutant, but not an otherwise isogenic wild-type strain, failed to develop starvation-induced thermotolerance or H2O2 resistance. The filamentous phenotype associated with DnaK deficiency was suppressed by cultivation in a defined glucose medium. When starved for glucose, the nonfilamentous and rod-shaped dnaK mutant strain failed to convert into the small spherical form typical of starving wild-type cells. The dnaK mutant retained the ability to develop adaptive H2O2 resistance during growth but not adaptive resistance to heat. Complementation of DnaK deficiency by using Ptac-regulated dnaK+ and dnaK+J+ expression plasmids confirmed a specific role for the DnaK molecular chaperone in these starvation-induced phenotypes.

A soluble maltase (alpha-glucosidase) with an apparent subunit mass of 80 kDa was purified to homogeneity from Sulfolobus solfataricus. The enzyme liberates glucose from maltose and malto-oligomers. Maximal activity was observed at 105 degrees C, with half-lives of 11 h (85 degrees C), 3.0 h (95 degrees C), and 2.75 h (100 degrees C). The enzyme was generally resistant to proteolysis and denaturants including aliphatic alcohols. n-Propanol treatment at 85 degrees C increased both Km and Vmax for maltose hydrolysis.

Archaea such asMetallosphaera sedulaare 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 forM. sedulato investigate parameters that limit bioleaching of chalcopyrite. The functional role of theM. sedula copRTAoperon was demonstrated by cross-species complementation of a copper-sensitiveSulfolobus solfataricus copRmutant. Inactivation of the gene encoding theM. sedulacopper efflux protein,copA, using targeted recombination compromised metal resistance and eliminated chalcopyrite bioleaching. In contrast, a spontaneousM. sedulamutant (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.