Paul Blum

Polypeptide flux through bacterial Hsp70: DnaK cooperates with trigger factor in chaperoning nascent chains.

A role for DnaK, the major E. coli Hsp70, in chaperoning de novo protein folding has remained elusive. Here we show that under nonstress conditions DnaK transiently associates with a wide variety of nascent and newly synthesized polypeptides, with a preference for chains larger than 30 kDa. Deletion of the nonessential gene encoding trigger factor, a ribosome-associated chaperone, results in a doubling of the fraction of nascent polypeptides interacting with DnaK. Combined deletion of the trigger factor and DnaK genes is lethal under normal growth conditions. These findings indicate important, partially overlapping functions of DnaK and trigger factor in de novo protein folding and explain why the loss of either chaperone can be tolerated by E. coli.

Occurrence and Characterization of Mercury Resistance in the Hyperthermophilic Archaeon Sulfolobus solfataricus by Use of Gene Disruption

Mercury resistance mediated by mercuric reductase (MerA) is widespread among bacteria and operates under the control of MerR. MerR represents a unique class of transcription factors that exert both positive and negative regulation on gene expression. Archaea and bacteria are prokaryotes, yet little is known about the biological role of mercury in archaea or whether a resistance mechanism occurs in these organisms. The archaeon Sulfolobus solfataricus was sensitive to mercuric chloride, and low-level adaptive resistance could be induced by metal preconditioning. Protein phylogenetic analysis of open reading frames SSO2689 and SSO2688 clarified their identity as orthologs of MerA and MerR. Northern analysis established that merA transcription responded to mercury challenge, since mRNA levels were transiently induced and, when normalized to 7S RNA, approximated values for other highly expressed transcripts. Primer extension analysis of merA mRNA predicted a noncanonical TATA box with nonstandard transcription start site spacing. The functional roles of merA and merR were clarified further by gene disruption. The merA mutant exhibited mercury sensitivity relative to wild type and was defective in elemental mercury volatilization, while the merR mutant was mercury resistant. Northern analysis of the merR mutant revealed merA transcription was constitutive and that transcript abundance was at maximum levels. These findings constitute the first report of an archaeal heavy metal resistance system; however, unlike bacteria the level of resistance is much lower. The archaeal system employs a divergent MerR protein that acts only as a negative transcriptional regulator of merA expression.

Targeted Disruption of the α-Amylase Gene in the Hyperthermophilic Archaeon Sulfolobus solfataricus

Sulfolobus solfataricus secretes an acid-resistant α-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 α-amylase function and regulation, the structural gene was identified and disrupted and the resulting mutant was characterized. Internal α-amylase peptide sequences obtained by tandem mass spectroscopy were used to identify the amyA coding sequence. Anti-α-amylase antibodies raised against the purified protein immunoprecipitated secreted α-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.

The Genome Sequence of the Metal-Mobilizing, Extremely Thermoacidophilic Archaeon Metallosphaera sedula Provides Insights into Bioleaching-Associated Metabolism

ABSTRACT 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.

Agmatidine, a modified cytidine in the anticodon of archaeal tRNA(Ile), base pairs with adenosine but not with guanosine.

Modification of the cytidine in the first anticodon position of the AUA decoding tRNAIle () 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 tRNAIle 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.

Flagellar Motility and Structure in the Hyperthermoacidophilic Archaeon Sulfolobus solfataricus

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.

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

Described is an efficient heterologous expression system for Sulfolobus solfataricus ADH-10 (Alcohol Dehydrogenase isozyme 10) and its use in the dynamic reductive kinetic resolution (DYRKR) of 2-arylpropanal (Profen-type) substrates. Importantly, among the 12 aldehydes tested, a general preference for the (S)-antipode was observed, with high ee’s for substrates corresponding to the NSAIDs (nonsteroidal anti-inflammatory drugs) naproxen, ibuprofen, flurbiprofen, ketoprofen, and fenoprofen. To our knowledge, this is the first application of a dehydrogenase from this Sulfolobus hyperthermophile to asymmetric synthesis and the first example of a DYRKR with such an enzyme. The requisite aldehydes are generated by Buchwald−Hartwig-type Pd(0)-mediated α-arylation of tert-butyl propionate. This is followed by reduction to the aldehyde in one [lithium diisobutyl tert-butoxyaluminum hydride (LDBBA)] or two steps [LAH/Dess−Martin periodinane]. Treatment of the profenal substrates with SsADH in 5% EtOH/phosphate buffer, pH 9, with catalytic NADH at 80 °C leads to efficient DYRKR, with ee’s exceeding 90% for 9 aryl side chains, including those of the aforementioned NSAIDs. An in silico model, consistent with the observed broad side chain tolerance, is presented. Importantly, the SsADH-10 enzyme could be conveniently recycled by exploiting the differential solubility of the organic substrate/product at 80 °C and at rt. Pleasingly, SsADH-10 could be taken through several “thermal cycles,” without erosion of ee, suggesting this as a generalizable approach to enzyme recycling for hyperthermophilic enzymes. Moreover, the robustness of this hyperthermophilic DH, in terms of both catalytic activity and stereochemical fidelity, speaks for greater examination of such archaeal enzymes in asymmetric synthesis.

Stability of mRNA in the hyperthermophilic archaeon Sulfolobus solfataricus

Archaea-like bacteria are prokaryotes but, in contrast, use eukaryotic-like systems for key aspects of DNA, RNA, and protein metabolism. mRNA is typically unstable in bacteria and stable in eukaryotes, but little information is available about mRNA half-lives in archaea. Because archaea are generally insensitive to antibiotics, examination of mRNA stability in the hyperthermophile, Sulfolobus solfataricus, required the identification of transcription inhibitors for half-life determinations. An improved lacS promoter-dependent in vitro transcription system was used to assess inhibitor action. Efficient inhibitors were distinguished as blocking both lacSp transcription in vitro and the incorporation of 3H-uracil into bulk RNA in vivo. Actinomycin D was the most stable and potent compound identified. A survey of transcript chemical half-lives normalized to levels of the signal recognition particle 7S RNA ranged from at least 2 h for tfb1, a transcription factor TFIIB paralog, to a minimum of 6.3 min for gln1, one of three glutamine synthetase paralogs. Transcript half-lives for other mRNAs were: 2 h, superoxide dismutase (sod); 37.5 min, glucose dehydrogenase (dhg1); 25 min, alpha-glucosidase (malA); and 13.5 min, transcription factor TFIIB-2 (tfb2) resulting in a minimum average half-life of 54 min. These are the first mRNA half-lives reported for a hyperthermophile or member of the crenarchaea. The unexpected stability of several transcripts has important implications for gene expression and mRNA degradation in this organism.

Regulation of Mercury Resistance in the Crenarchaeote Sulfolobus solfataricus

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.

Something Old, Something New, Something Borrowed; How the Thermoacidophilic Archaeon Sulfolobus solfataricus Responds to Oxidative Stress

To avoid molecular damage of biomolecules due to oxidation, all cells have evolved constitutive and responsive systems to mitigate and repair chemical modifications. Archaea have adapted to some of the most extreme environments known to support life, including highly oxidizing conditions. However, in comparison to bacteria and eukaryotes, relatively little is known about the biology and biochemistry of archaea in response to changing conditions and repair of oxidative damage. In this study transcriptome, proteome, and chemical reactivity analyses of hydrogen peroxide (H2O2) induced oxidative stress in Sulfolobus solfataricus (P2) were conducted. Microarray analysis of mRNA expression showed that 102 transcripts were regulated by at least 1.5 fold, 30 minutes after exposure to 30 µM H2O2. Parallel proteomic analyses using two-dimensional differential gel electrophoresis (2D-DIGE), monitored more than 800 proteins 30 and 105 minutes after exposure and found that 18 had significant changes in abundance. A recently characterized ferritin-like antioxidant protein, DPSL, was the most highly regulated species of mRNA and protein, in addition to being post-translationally modified. As expected, a number of antioxidant related mRNAs and proteins were differentially regulated. Three of these, DPSL, superoxide dismutase, and peroxiredoxin were shown to interact and likely form a novel supramolecular complex for mitigating oxidative damage. A scheme for the ability of this complex to perform multi-step reactions is presented. Despite the central role played by DPSL, cells maintained a lower level of protection after disruption of the dpsl gene, indicating a level of redundancy in the oxidative stress pathways of S. solfataricus. This work provides the first “omics” scale assessment of the oxidative stress response for an archeal organism and together with a network analysis using data from previous studies on bacteria and eukaryotes reveals evolutionarily conserved pathways where complex and overlapping defense mechanisms protect against oxygen toxicity.