An ArsR/SmtB family member is involved in the regulation by arsenic of the arsenite oxidase operon in Thiomonas arsenitoxydans.

The genetic organization of the aioBA operon, encoding the arsenite oxidase of the moderately acidophilic and facultative chemoautotrophic bacterium Thiomonas arsenitoxydans, is different from that of the aioBA operon in the other arsenite oxidizers, in that it encodes AioF, a metalloprotein belonging to the ArsR/SmtB family. AioF is stabilized by arsenite, arsenate, or antimonite but not molybdate. Arsenic is tightly attached to AioF, likely by cysteine residues. When loaded with arsenite or arsenate, AioF is able to bind specifically to the regulatory region of the aio operon at two distinct positions. In Thiomonas arsenitoxydans, the promoters of aioX and aioB are convergent, suggesting that transcriptional interference occurs. These results indicate that the regulation of the aioBA operon is more complex in Thiomonas arsenitoxydans than in the other aioBA containing arsenite oxidizers and that the arsenic binding protein AioF is involved in this regulation. On the basis of these data, a model to explain the tight control of aioBA expression by arsenic in Thiomonas arsenitoxydans is proposed.

Organization and regulation of the arsenite oxidase operon of the moderately acidophilic and facultative chemoautotrophic Thiomonas arsenitoxydans.

Thiomonas arsenitoxydans is an acidophilic and facultatively autotrophic bacterium that can grow by oxidizing arsenite to arsenate. A comparative genomic analysis showed that the T. arsenitoxydans aioBA cluster encoding the two subunits of arsenite oxidase is distinct from the other clusters, with two specific genes encoding a cytochrome c and a metalloregulator belonging to the ArsR/SmtB family. These genes are cotranscribed with aioBA, suggesting that these cytochromes c are involved in arsenite oxidation and that this operon is controlled by the metalloregulator. The growth of T. arsenitoxydans in the presence of thiosulfate and arsenite, or arsenate, is biphasic. Real-time PCR experiments showed that the operon is transcribed during the second growth phase in the presence of arsenite or arsenate, whereas antimonite had no effect. These results suggest that the expression of the aioBA operon of T. arsenitoxydans is regulated by the electron donor present in the medium, i.e., is induced in the presence of arsenic but is repressed by more energetic substrates. Our data indicate that the genetic organization and regulation of the aioBA operon of T. arsenitoxydans differ from those of the other arsenite oxidizers.

Characteristics of a phylogenetically ambiguous, arsenic-oxidizing Thiomonas sp., Thiomonas arsenitoxydans strain 3As(T) sp. nov.

A moderately acidophilic, facultative chemoautotrophic, As(III)-oxidizing Thiomonas sp. (strain 3As(T)) was previously shown, on the basis of comparative 16S rRNA gene sequences, to be closely related to both Tm. perometabolis DSM 18570(T) and Tm. intermedia DSM 18155(T). While it had shared many physiological traits with Tm. intermedia (T), a mean DNA-DNA hybridization value (DDHV) of 47.2% confirmed that strain 3As(T) was not a strain of Tm. intermedia, though the situation with regard to Tm. perometabolis (DDHV previously determined as 72%) was more ambiguous. A comparative physiological and chemotaxonomic study of strain 3As(T) and Tm. perometabolis (T) was therefore carried out, together with multilocus sequence analysis (MLSA) of all three bacteria. Differences in fatty acid profiles and utilization of organic substrates supported the view that strain 3As(T) and Tm. perometabolis are distinct species, while MLSA showed a closer relationship between strain 3As(T) and Tm. intermedia (T) than between strain 3As(T) and Tm. perometabolis (T). These apparent contradictory conclusions were explained by differences in genome of these three bacteria, which are known to be highly flexible in Thiomonas spp. A novel species designation Thiomonas arsenitoxydans is proposed for strain 3As(T) (DSM 22701(T), CIP 110005(T)), which is nominated as the type strain of this species.

Existence of abnormal protein aggregates in healthy Escherichia coli cells.

Protein aggregation is a phenomenon observed in all organisms and has often been linked with cell disorders. In addition, several groups have reported a virtual absence of protein aggregates in healthy cells. In contrast to previous studies and the expected outcome, we observed aggregated proteins in aerobic exponentially growing and "healthy" Escherichia coli cells. We observed overrepresentation of "aberrant proteins," as well as substrates of the major conserved chaperone DnaK (Hsp70) and the protease ClpXP (a serine protease), in the aggregates. In addition, the protein aggregates appeared to interact with chaperones known to be involved in the aggregate repair pathway, including ClpB, GroEL, GroES, and DnaK. Finally, we showed that the levels of reactive oxygen species and unfolded or misfolded proteins determine the levels of protein aggregates. Our results led us to speculate that protein aggregates may function as a temporary "trash organelle" for cellular detoxification.

Unexpected altered specificity is responsible for St. Louis encephalitis virus recombinant protease autoproteolysis.

We report herein the study of the cleavage fragments generated by autoproteolysis of the St. Louis encephalitis virus recombinant protease. The cleavage sites leading to truncated forms were identified by microsequencing, which revealed an unexpected altered specificity of the recombinant proteinase towards unusual sequences.

The Global Redox Responding RegB/RegA Signal Transduction System Regulates the Genes Involved in Ferrous Iron and Inorganic Sulfur Compound Oxidation of the Acidophilic Acidithiobacillus ferrooxidans.

The chemical attack of ore by ferric iron and/or sulfuric acid releases valuable metals. The products of these reactions are recycled by iron and sulfur oxidizing microorganisms. These acidophilic chemolithotrophic prokaryotes, among which Acidithiobacillus ferrooxidans, grow at the expense of the energy released from the oxidation of ferrous iron and/or inorganic sulfur compounds (ISCs). In At. ferrooxidans, it has been shown that the expression of the genes encoding the proteins involved in these respiratory pathways is dependent on the electron donor and that the genes involved in iron oxidation are expressed before those responsible for ISCs oxidation when both iron and sulfur are present. Since the redox potential increases during iron oxidation but remains stable during sulfur oxidation, we have put forward the hypothesis that the global redox responding two components system RegB/RegA is involved in this regulation. To understand the mechanism of this system and its role in the regulation of the aerobic respiratory pathways in At. ferrooxidans, the binding of different forms of RegA (DNA binding domain, wild-type, unphosphorylated and phosphorylated-like forms of RegA) on the regulatory region of different genes/operons involved in ferrous iron and ISC oxidation has been analyzed. We have shown that the four RegA forms are able to bind specifically the upstream region of these genes. Interestingly, the phosphorylation of RegA did not change its affinity for its cognate DNA. The transcriptional start site of these genes/operons has been determined. In most cases, the RegA binding site(s) was (were) located upstream from the -35 (or -24) box suggesting that RegA does not interfere with the RNA polymerase binding. Based on the results presented in this report, the role of the RegB/RegA system in the regulation of the ferrous iron and ISC oxidation pathways in At. ferrooxidans is discussed.

Response of the anaerobe Desulfovibrio vulgaris Hildenborough to oxidative conditions: proteome and transcript analysis.

The method of two-dimensional protein gel electrophoresis was used to evaluate the changes at the proteins level following oxygen exposure of the anaerobic sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough. Fifty-seven proteins showed significant differential expression. The cellular concentration of 35 proteins decreased while that of nineteen increased as a specific consequence of oxidative conditions. The proteins that were less abundant belonged to various functional categories such as nucleic acid and protein biosynthesis, detoxification mechanisms, or cell division. Interestingly, quantitative real-time PCR revealed that the genes encoding detoxification enzymes (rubrerythrins, superoxide reductase) are down regulated. The loss of viability of D. vulgaris Hildenborough under these oxidative conditions (Fournier et al., J. Biol. Chem. 279 (2004) 1785) can be directly related to the decrease in the cellular concentrations of these proteins, thereby specifying the toxicity of oxygen for the cells. Among the proteins that were more abundant under oxygen exposure, several thiol-specific peroxidases (thiol-peroxidase, BCP-like protein, and putative glutaredoxin) were identified. Using RT-PCR, the up-regulation of the genes encoding the thiol-peroxidase and the BCP was demonstrated. That is the first time that these proteins have been shown to be involved in the defense of D. vulgaris toward an oxidative stress. Several hypothetical proteins were also shown to be differentially expressed. A function in the defense mechanism against an oxidative stress is proposed for these uncharacterized proteins.

Insights into the Quorum Sensing Regulon of the Acidophilic Acidithiobacillus ferrooxidans Revealed by Transcriptomic in the Presence of an Acyl Homoserine Lactone Superagonist Analog.

While a functional quorum sensing system has been identified in the acidophilic chemolithoautotrophic Acidithiobacillus ferrooxidans ATCC 23270(T) and shown to modulate cell adhesion to solid substrates, nothing is known about the genes it regulates. To address the question of how quorum sensing controls biofilm formation in A. ferrooxidans (T), the transcriptome of this organism in conditions in which quorum sensing response is stimulated by a synthetic superagonist AHL (N-acyl homoserine lactones) analog has been studied. First, the effect on biofilm formation of a synthetic AHL tetrazolic analog, tetrazole 9c, known for its agonistic QS activity, was assessed by fluorescence and electron microscopy. A fast adherence of A. ferrooxidans (T) cells on sulfur coupons was observed. Then, tetrazole 9c was used in DNA microarray experiments that allowed the identification of genes regulated by quorum sensing signaling, and more particularly, those involved in early biofilm formation. Interestingly, afeI gene, encoding the AHL synthase, but not the A. ferrooxidans quorum sensing transcriptional regulator AfeR encoding gene, was shown to be regulated by quorum sensing. Data indicated that quorum sensing network represents at least 4.5% (141 genes) of the ATCC 23270(T) genome of which 42.5% (60 genes) are related to biofilm formation. Finally, AfeR was shown to bind specifically to the regulatory region of the afeI gene at the level of the palindromic sequence predicted to be the AfeR binding site. Our results give new insights on the response of A. ferrooxidans to quorum sensing and on biofilm biogenesis.

New insights into the respiratory chains of the chemolithoautotrophic and hyperthermophilic bacterium Aquifex aeolicus.

Aquifex aeolicus, a highly hyperthermophilic bacterium, grows chemolithoautotrophically at 85 degrees C, with hydrogen as electron donor and oxygen as electron acceptor in the presence of a sulfur compound. Stimulated by its exceptional physiological properties, we have set out to study the oxygen metabolism of this microorganism. With the use of an unconventional integrative proteomic approach combining separation of membrane proteins by Blue-Native electrophoresis, detection of enzyme activities in-gel and direct protein identification by two-dimensional liquid chromatography and tandem mass spectrometry (2D nanoLC-MS/MS), we have obtained evidence for the presence of functional respiratory enzymes in membranes of A. aeolicus cultivated with H2/O2/S0 as well as an organization in stable superstructures of some of these individual complexes. This study has revealed the assembly of the bc complex and a cytochrome coxidase as a supercomplex and possible associations of electron transfer proteins and complexes involved in oxygen reduction such as sulfide quinone reductase, cytochrome c oxidase, bc complex, membrane-bound hydrogenase I and quinol oxidase. Electron transfer measurements on solubilized membranes have demonstrated the existence of uncommon respiratory chains (sulfide/oxygen as well as hydrogen/oxygen) in the cell growth conditions used. Moreover, the subunit composition of some of the complexes has been more precisely described, particularly that of complex I, leading for the first time to evidence of the presence of several isoforms of this complex. We can propose from our results (in-gel identification and functional data) that the bioenergetic pathways (sulfur and oxygen reductions) may be organized in supramolecular structures in A. aeolicus, as we have previously purified and characterized a hydrogen-oxidizing sulfur-reducing supercomplex from this bacterium.

Proteome analysis of Rickettsia felis highlights the expression profile of intracellular bacteria.

The proteome of Rickettsia felis, an obligate intracellular bacterium responsible for spotted fever, was analyzed using two complementary proteomic approaches: 2-DE coupled with MALDI-TOF, and SDS-PAGE with nanoLC-MS/MS. This strategy allowed identification of 165 proteins and helped to answer some questions raised by the genome sequence of this bacterium. We successfully identified potential virulence factors including two putative adhesins, four proteins of the type IV secretion system, four Sca autotransporters, four components of ABC transporters, some R. felis-specific proteins, and one antitoxin of the toxin-antitoxin system. Notably, the antitoxin was the first to be identified in intracellular bacteria. Only one protein containing rickettsia palindromic repeats was found, whereas none of the split genes, transposases, or tetratricopeptide/ankyrin repeats were detectably expressed. Comparison of the protein expression profiles of R. felis and 23 other bacterial species according to functional categories showed that intracellular bacteria express more proteins related to translation, especially ribosomal proteins. However, the remaining bacteria express more proteins related to energy production and carbohydrate/amino acid metabolism. In conclusion, this study reveals R. felis virulence factor expression and highlights the unique protein expression profile of intracellular bacteria.

Mimivirus giant particles incorporate a large fraction of anonymous and unique gene products.

Acanthamoeba polyphaga mimivirus is the largest known virus in both particle size and genome complexity. Its 1.2-Mb genome encodes 911 proteins, among which only 298 have predicted functions. The composition of purified isolated virions was analyzed by using a combined electrophoresis/mass spectrometry approach allowing the identification of 114 proteins. Besides the expected major structural components, the viral particle packages 12 proteins unambiguously associated with transcriptional machinery, 3 proteins associated with DNA repair, and 2 topoisomerases. Other main functional categories represented in the virion include oxidative pathways and protein modification. More than half of the identified virion-associated proteins correspond to anonymous genes of unknown function, including 45 "ORFans." As demonstrated by both Western blotting and immunogold staining, some of these "ORFans," which lack any convincing similarity in the sequence databases, are endowed with antigenic properties. Thus, anonymous and unique genes constituting the majority of the mimivirus gene complement encode bona fide proteins that are likely to participate in well-integrated processes.