Interplay between transcriptional regulators and VapBC toxin-antitoxin loci during thermal stress response in extremely thermoacidophilic archaea.

Thermoacidophilic archaea lack sigma factors and the large inventory of heat shock proteins (HSPs) widespread in bacterial genomes, suggesting other strategies for handling thermal stress are involved. Heat shock transcriptomes for the thermoacidophilic archaeon Saccharolobus (f. Sulfolobus) solfataricus 98/2 revealed genes that were highly responsive to thermal stress, including transcriptional regulators YtrASs (Ssol_2420) and FadRSs (Ssol_0314), as well as type II toxin-antitoxin (TA) loci VapBC6 (Ssol_2337, Ssol_2338) and VapBC22 (Ssol_0819, Ssol_0818). The role, if any, of type II TA loci during stress response in microorganisms, such as Escherichia coli, is controversial. But, when genes encoding YtrASs , FadRSs , VapC22, VapB6, and VapC6 were systematically mutated in Sa. solfataricus 98/2, significant up-regulation of the other genes within this set was observed, implicating an interconnected regulatory network during thermal stress response. VapBC6 and VapBC22 have close homologues in other Sulfolobales, as well as in other archaea (e.g. Pyrococcus furiosus and Archaeoglobus fulgidus), and their corresponding genes were also heat shock responsive. The interplay between VapBC TA loci and heat shock regulators in Sa solfataricus 98/2 not only indicates a cellular mechanism for heat shock response that differs from bacteria but one that could have common features within the thermophilic archaea.

Retargeting the Clostridium botulinum C2 toxin to the neuronal cytosol.

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.

Uranium extremophily is an adaptive, rather than intrinsic, feature for extremely thermoacidophilic Metallosphaera species.

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.

Role of vapBC toxin-antitoxin loci in the thermal stress response of Sulfolobus solfataricus.

TA (toxin-antitoxin) loci are ubiquitous in prokaryotic micro-organisms, including archaea, yet their physiological function is largely unknown. For example, preliminary reports have suggested that TA loci are microbial stress-response elements, although it was recently shown that knocking out all known chromosomally located TA loci in Escherichia coli did not have an impact on survival under certain types of stress. The hyperthermophilic crenarchaeon Sulfolobus solfataricus encodes at least 26 vapBC (where vap is virulence-associated protein) family TA loci in its genome. VapCs are PIN (PilT N-terminus) domain proteins with putative ribonuclease activity, while VapBs are proteolytically labile proteins, which purportedly function to silence VapCs when associated as a cognate pair. Global transcriptional analysis of S. solfataricus heat-shock-response dynamics (temperature shift from 80 to 90 degrees C) revealed that several vapBC genes were triggered by the thermal shift, suggesting a role in heat-shock-response. Indeed, knocking out a specific vapBC locus in S. solfataricus substantially changed the transcriptome and, in one case, rendered the crenarchaeon heat-shock-labile. These findings indicate that more work needs to be done to determine the role of VapBCs in S. solfataricus and other thermophilic archaea, especially with respect to post-transcriptional regulation.

Plasmid transformation and expression of the firefly luciferase in Microbacterium testaceum type and endophytic colonizing field strains.

Microbacterium testaceum is a predominant endophytic bacterial species isolated from corn and sorghum in the midwestern United States. The development of genetic transfer systems for M. testaceum may enable its use for biocontrol and other applications. The type strain (IFO 12675) and field isolates (SE017, SE034, and CE648) were grown to mid-exponential phase, concentrated (1.0 x 1011 CFU x mL(-1)), electroporated (Escherichia coli-Clavibacter shuttle plasmid pDM302), and plated on TSA with 10 microg x mL(-1) chloramphenicol. Transformation efficiencies averaged 140 CFU x microg(-1) of DNA. Restriction endonuclease analysis showed that pDM302 was not altered after extraction from transformants and re-introduction into E. coli. Transformants with pDM302 were also subjected to nonselective growth conditions, with the frequency of loss after one passage being 84% for IFO 12675 and 88% for SE034. We inserted the green fluorescent protein and the firefly luciferase (FFlux) reporter genes into pDM302, confirming the expression of FFlux in IFO 12675 and SE034. The SE034 FFlux strain was recovered from inoculated corn in greenhouse studies and found to fluoresce by luminometry. These results in M. testaceum demonstrate for the first time its transformability, pDM302 replication, FFlux gene expression, and the recovery of the FFlux recombinant strain from inoculated corn.

The genome sequence of the metal-mobilizing, extremely thermoacidophilic archaeon Metallosphaera sedula 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, approximately 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.