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1.  Stone-dwelling actinobacteria Blastococcus saxobsidens, Modestobacter marinus and Geodermatophilus obscurus proteogenomes 
The ISME Journal  2015;10(1):21-29.
The Geodermatophilaceae are unique model systems to study the ability to thrive on or within stones and their proteogenomes (referring to the whole protein arsenal encoded by the genome) could provide important insight into their adaptation mechanisms. Here we report the detailed comparative genome analysis of Blastococcus saxobsidens (Bs), Modestobacter marinus (Mm) and Geodermatophilus obscurus (Go) isolated respectively from the interior and the surface of calcarenite stones and from desert sandy soils. The genome-scale analysis of Bs, Mm and Go illustrates how adaptation to these niches can be achieved through various strategies including ‘molecular tinkering/opportunism' as shown by the high proportion of lost, duplicated or horizontally transferred genes and ORFans. Using high-throughput discovery proteomics, the three proteomes under unstressed conditions were analyzed, highlighting the most abundant biomarkers and the main protein factors. Proteomic data corroborated previously demonstrated stone-related ecological distribution. For instance, these data showed starvation-inducible, biofilm-related and DNA-protection proteins as signatures of the microbes associated with the interior, surface and outside of stones, respectively.
doi:10.1038/ismej.2015.108
PMCID: PMC4681853  PMID: 26125681
2.  Essentiality of threonylcarbamoyladenosine (t6A), a universal tRNA modification, in bacteria 
Molecular microbiology  2015;98(6):1199-1221.
Threonylcarbamoyladenosine (t6A) is a modified nucleoside universally conserved in tRNAs in all three kingdoms of life. The recently discovered genes for t6A synthesis, including tsaC and tsaD, are essential in model prokaryotes but not essential in yeast. These genes had been identified as antibacterial targets even before their functions were known. However, the molecular basis for this prokaryotic-specific essentiality has remained a mystery. Here, we show that t6A is a strong positive determinant for aminoacylation of tRNA by bacterial-type but not by eukaryotic-type isoleucyl-tRNA synthetases and might also be a determinant for the essential enzyme tRNAIle-lysidine synthetase. We confirm that t6A is essential in Escherichia coli and a survey of genome-wide essentiality studies shows that genes for t6A synthesis are essential in most prokaryotes. This essentiality phenotype is not universal in Bacteria as t6A is dispensable in Deinococcus radiodurans, Thermus thermophilus, Synechocystis PCC6803 and Streptococcus mutans. Proteomic analysis of t6A- D. radiodurans strains revealed an induction of the proteotoxic stress response and identified genes whose translation is most affected by the absence of t6A in tRNAs. Thus, although t6A is universally conserved in tRNAs, its role in translation might vary greatly between organisms.
doi:10.1111/mmi.13209
PMCID: PMC4963248  PMID: 26337258
tRNA; maturation; translation; modified nucleosides; t6A
3.  Ovary and embryo proteogenomic dataset revealing diversity of vitellogenins in the crustacean Gammarus fossarum 
Data in Brief  2016;8:1259-1262.
Ovaries and embryos from sexually mature Gammarus fossarum were sampled at different stages of the reproductive cycle. The soluble proteome was extracted for five biological replicates and samples were subjected to trypsin digestion. The resulting peptides were analyzed by high resolution tandem mass spectrometry with a LTQ-Orbitrap XL instrument. The MS/MS spectra were assigned with a previously described RNAseq-derived G. fossarum database. The proteins highlighted by proteogenomics were monitored and their abundance kinetics over the different stages revealed a large panel of vitellogenins. Criteria were i) accumulation during oogenesis, ii) decrease during embryogenesis, iii) classified as female-specific, and iv) sequence similarity and phylogenetic analysis. The data accompanying the manuscript describing the database searches and comparative analysis (“High-throughput proteome dynamics for discovery of key proteins in sentinel species: unsuspected vitellogenins diversity in the crustacean Gammarus fossarum” by Trapp et al. [1]) have been deposited to the ProteomeXchange via the PRIDE repository with identifiers PRIDE: PXD001002.
doi:10.1016/j.dib.2016.07.045
PMCID: PMC4983104  PMID: 27547807
Crustacean; Embryogenesis; Non-model organism; Oogenesis; Proteogenomics Reproduction; Vitellogenins
4.  Proteome data to explore the impact of pBClin15 on Bacillus cereus ATCC 14579 
Data in Brief  2016;8:1243-1246.
This data article reports changes in the cellular and exoproteome of B. cereus cured from pBClin15.Time-course changes of proteins were assessed by high-throughput nanoLC-MS/MS. We report all the peptides and proteins identified and quantified in B. cereus with and without pBClin15. Proteins were classified into functional groups using the information available in the KEGG classification and we reported their abundance in term of normalized spectral abundance factor. The repertoire of experimentally confirmed proteins of B. cereus presented here is the largest ever reported, and provides new insights into the interplay between pBClin15 and its host B. cereus ATCC 14579. The data reported here is related to a published shotgun proteomics analysis regarding the role of pBClin15, “Deciphering the interactions between the Bacillus cereus linear plasmid, pBClin15, and its host by high-throughput comparative proteomics” Madeira et al. [1]. All the associated mass spectrometry data have been deposited in the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository (http://www.ebi.ac.uk/pride/), with the dataset identifier PRIDE: PXD001568, PRIDE: PXD002788 and PRIDE: PXD002789.
doi:10.1016/j.dib.2016.07.042
PMCID: PMC4983103  PMID: 27547804
5.  “You produce while I clean up”, a strategy revealed by exoproteomics during Synechococcus–Roseobacter interactions 
Proteomics  2015;15(20):3454-3462.
Most of the energy that is introduced into the oceans by photosynthetic primary producers is in the form of organic matter that then sustains the rest of the food web, from micro to macro‐organisms. However, it is the interactions between phototrophs and heterotrophs that are vital to maintaining the nutrient balance of marine microbiomes that ultimately feed these higher trophic levels. The primary produced organic matter is mostly remineralized by heterotrophic microorganisms but, because most of the oceanic dissolved organic matter is in the form of biopolymers, and microbial membrane transport systems operate with molecules <0.6 kDa, it must be hydrolyzed outside the cell before a microorganism can acquire it. As a simili of the marine microbiome, we analyzed, using state‐of‐the‐art proteomics, the exoproteomes obtained from synthetic communities combining specific Roseobacter (Ruegeria pomeroyi DSS‐3, Roseobacter denitrificans OCh114, and Dinoroseobacter shibae DFL‐12) and Synechococcus strains (WH7803 and WH8102). This approach identified the repertoire of hydrolytic enzymes secreted by Roseobacter, opening up the black box of heterotrophic transformation/remineralization of biopolymers generated by marine phytoplankton. As well as highlighting interesting exoenzymes this strategy also allowed us to infer clues on the molecular basis of niche partitioning.
doi:10.1002/pmic.201400562
PMCID: PMC4949626  PMID: 25728650
Dissolved organic matter; Exoproteome; Marine microbial interactions; Microbiology; Roseobacter; Secreted enzymes
6.  Functional distinctness in the exoproteomes of marine S ynechococcus  
Environmental Microbiology  2015;17(10):3781-3794.
Summary
The exported protein fraction of an organism may reflect its life strategy and, ultimately, the way it is perceived by the outside world. Bioinformatic prediction of the exported pan‐proteome of P rochlorococcus and S ynechococcus lineages demonstrated that (i) this fraction of the encoded proteome had a much higher incidence of lineage‐specific proteins than the cytosolic fraction (57% and 73% homologue incidence respectively) and (ii) exported proteins are largely uncharacterized to date (54%) compared with proteins from the cytosolic fraction (35%). This suggests that the genomic and functional diversity of these organisms lies largely in the diverse pool of novel functions these organisms export to/through their membranes playing a key role in community diversification, e.g. for niche partitioning or evading predation. Experimental exoproteome analysis of marine S ynechococcus showed transport systems for inorganic nutrients, an interesting array of strain‐specific exoproteins involved in mutualistic or hostile interactions (i.e. hemolysins, pilins, adhesins), and exoenzymes with a potential mixotrophic goal (i.e. exoproteases and chitinases). We also show how these organisms can remodel their exoproteome, i.e. by increasing the repertoire of interaction proteins when grown in the presence of a heterotroph or decrease exposure to prey when grown in the dark. Finally, our data indicate that heterotrophic bacteria can feed on the exoproteome of S ynechococcus.
doi:10.1111/1462-2920.12822
PMCID: PMC4949707  PMID: 25727668
7.  PprA Protein Is Involved in Chromosome Segregation via Its Physical and Functional Interaction with DNA Gyrase in Irradiated Deinococcus radiodurans Bacteria 
mSphere  2016;1(1):e00036-15.
D. radiodurans is one of the most radiation-resistant organisms known. This bacterium is able to cope with high levels of DNA lesions generated by exposure to extreme doses of ionizing radiation and to reconstruct a functional genome from hundreds of radiation-induced chromosomal fragments. Here, we identified partners of PprA, a radiation-induced Deinococcus-specific protein, previously shown to be required for radioresistance. Our study leads to three main findings: (i) PprA interacts with DNA gyrase after irradiation, (ii) treatment of cells with novobiocin results in defects in chromosome segregation that are aggravated by the absence of PprA, and (iii) PprA stimulates the decatenation activity of DNA gyrase. Our results extend the knowledge of how D. radiodurans cells survive exposure to extreme doses of gamma irradiation and point out the link between DNA repair, chromosome segregation, and DNA gyrase activities in the radioresistant D. radiodurans bacterium.
ABSTRACT
PprA, a radiation-induced Deinococcus-specific protein, was previously shown to be required for cell survival and accurate chromosome segregation after exposure to ionizing radiation. Here, we used an in vivo approach to determine, by shotgun proteomics, putative PprA partners coimmunoprecipitating with PprA when cells were exposed to gamma rays. Among them, we found the two subunits of DNA gyrase and, thus, chose to focus our work on characterizing the activities of the deinococcal DNA gyrase in the presence or absence of PprA. Loss of PprA rendered cells hypersensitive to novobiocin, an inhibitor of the B subunit of DNA gyrase. We showed that treatment of bacteria with novobiocin resulted in induction of the radiation desiccation response (RDR) regulon and in defects in chromosome segregation that were aggravated by the absence of PprA. In vitro, the deinococcal DNA gyrase, like other bacterial DNA gyrases, possesses DNA negative supercoiling and decatenation activities. These two activities are inhibited in vitro by novobiocin and nalidixic acid, whereas PprA specifically stimulates the decatenation activity of DNA gyrase. Together, these results suggest that PprA plays a major role in chromosome decatenation via its interaction with the deinococcal DNA gyrase when D. radiodurans cells are recovering from exposure to ionizing radiation.
IMPORTANCE D. radiodurans is one of the most radiation-resistant organisms known. This bacterium is able to cope with high levels of DNA lesions generated by exposure to extreme doses of ionizing radiation and to reconstruct a functional genome from hundreds of radiation-induced chromosomal fragments. Here, we identified partners of PprA, a radiation-induced Deinococcus-specific protein, previously shown to be required for radioresistance. Our study leads to three main findings: (i) PprA interacts with DNA gyrase after irradiation, (ii) treatment of cells with novobiocin results in defects in chromosome segregation that are aggravated by the absence of PprA, and (iii) PprA stimulates the decatenation activity of DNA gyrase. Our results extend the knowledge of how D. radiodurans cells survive exposure to extreme doses of gamma irradiation and point out the link between DNA repair, chromosome segregation, and DNA gyrase activities in the radioresistant D. radiodurans bacterium.
doi:10.1128/mSphere.00036-15
PMCID: PMC4863600  PMID: 27303692
Deinococcus radiodurans; PprA; DNA gyrase; DNA decatenation
8.  Implementation of meiosis prophase I programme requires a conserved retinoid-independent stabilizer of meiotic transcripts 
Nature Communications  2016;7:10324.
Sexual reproduction is crucially dependent on meiosis, a conserved, specialized cell division programme that is essential for the production of haploid gametes. Here we demonstrate that fertility and the implementation of the meiotic programme require a previously uncharacterized meiosis-specific protein, MEIOC. Meioc invalidation in mice induces early and pleiotropic meiotic defects in males and females. MEIOC prevents meiotic transcript degradation and interacts with an RNA helicase that binds numerous meiotic mRNAs. Our results indicate that proper engagement into meiosis necessitates the specific stabilization of meiotic transcripts, a previously little-appreciated feature in mammals. Remarkably, the upregulation of MEIOC at the onset of meiosis does not require retinoic acid and STRA8 signalling. Thus, we propose that the complete induction of the meiotic programme requires both retinoic acid-dependent and -independent mechanisms. The latter process involving post-transcriptional regulation likely represents an ancestral mechanism, given that MEIOC homologues are conserved throughout multicellular animals.
Meiosis is a cell division program that produces haploid gametes and is initiated by a retinoic acid-dependent process. Here the authors report that a meiosis-specific protein, MEIOC, is upregulated in a retinoic acid-independent manner and is required to stabilise meiosis-specific transcripts.
doi:10.1038/ncomms10324
PMCID: PMC4729902  PMID: 26742488
9.  Understanding butanol tolerance and assimilation in P seudomonas putida  BIRD‐1: an integrated omics approach 
Microbial Biotechnology  2016;9(1):100-115.
Summary
P seudomonas putida  BIRD‐1 has the potential to be used for the industrial production of butanol due to its solvent tolerance and ability to metabolize low‐cost compounds. However, the strain has two major limitations: it assimilates butanol as sole carbon source and butanol concentrations above 1% (v/v) are toxic. With the aim of facilitating BIRD‐1 strain design for industrial use, a genome‐wide mini‐Tn5 transposon mutant library was screened for clones exhibiting increased butanol sensitivity or deficiency in butanol assimilation. Twenty‐one mutants were selected that were affected in one or both of the processes. These mutants exhibited insertions in various genes, including those involved in the TCA cycle, fatty acid metabolism, transcription, cofactor synthesis and membrane integrity. An omics‐based analysis revealed key genes involved in the butanol response. Transcriptomic and proteomic studies were carried out to compare short and long‐term tolerance and assimilation traits. P seudomonas putida initiates various butanol assimilation pathways via alcohol and aldehyde dehydrogenases that channel the compound to central metabolism through the glyoxylate shunt pathway. Accordingly, isocitrate lyase – a key enzyme of the pathway – was the most abundant protein when butanol was used as the sole carbon source. Upregulation of two genes encoding proteins PPUBIRD1_2240 and PPUBIRD1_2241 (acyl‐CoA dehydrogenase and acyl‐CoA synthetase respectively) linked butanol assimilation with acyl‐CoA metabolism. Butanol tolerance was found to be primarily linked to classic solvent defense mechanisms, such as efflux pumps, membrane modifications and control of redox state. Our results also highlight the intensive energy requirements for butanol production and tolerance; thus, enhancing TCA cycle operation may represent a promising strategy for enhanced butanol production.
doi:10.1111/1751-7915.12328
PMCID: PMC4720416  PMID: 26986205
10.  Proteomics identifies Bacillus cereus EntD as a pivotal protein for the production of numerous virulence factors 
Bacillus cereus is a Gram-positive pathogen that causes a wide variety of diseases in humans. It secretes into the extracellular milieu proteins that may contribute directly or indirectly to its virulence. EntD is a novel exoprotein identified by proteogenomics of B. cereus ATCC 14579. We constructed a ΔentD mutant and analyzed the impact of entD disruption on the cellular proteome and exoproteome isolated from early, late, and stationary-phase cultures. We identified 308 and 79 proteins regulated by EntD in the cellular proteome and the exoproteome, respectively. The contribution of these proteins to important virulence-associated functions, including central metabolism, cell structure, antioxidative ability, cell motility, and toxin production, are presented. The proteomic data were correlated with the growth defect, cell morphology change, reduced motility, and reduced cytotoxicity of the ΔentD mutant strain. We conclude that EntD is an important player in B. cereus virulence. The function of EntD and the putative EntD-dependent regulatory network are discussed. To our knowledge, this study is the first characterization of an Ent family protein in a species of the B. cereus group.
doi:10.3389/fmicb.2015.01004
PMCID: PMC4595770  PMID: 26500610
comparative proteomics; cellular proteome; exoproteome; Bacillus cereus; metabolism; virulence
11.  Salt Stress Induced Changes in the Exoproteome of the Halotolerant Bacterium Tistlia consotensis Deciphered by Proteogenomics 
PLoS ONE  2015;10(8):e0135065.
The ability of bacteria to adapt to external osmotic changes is fundamental for their survival. Halotolerant microorganisms, such as Tistlia consotensis, have to cope with continuous fluctuations in the salinity of their natural environments which require effective adaptation strategies against salt stress. Changes of extracellular protein profiles from Tistlia consotensis in conditions of low and high salinities were monitored by proteogenomics using a bacterial draft genome. At low salinity, we detected greater amounts of the HpnM protein which is involved in the biosynthesis of hopanoids. This may represent a novel, and previously unreported, strategy by halotolerant microorganisms to prevent the entry of water into the cell under conditions of low salinity. At high salinity, proteins associated with osmosensing, exclusion of Na+ and transport of compatible solutes, such as glycine betaine or proline are abundant. We also found that, probably in response to the high salt concentration, T. consotensis activated the synthesis of flagella and triggered a chemotactic response neither of which were observed at the salt concentration which is optimal for growth. Our study demonstrates that the exoproteome is an appropriate indicator of adaptive response of T. consotensis to changes in salinity because it allowed the identification of key proteins within its osmoadaptive mechanism that had not previously been detected in its cell proteome.
doi:10.1371/journal.pone.0135065
PMCID: PMC4545795  PMID: 26287734
12.  Data for comparative proteomics of ovaries from five non-model, crustacean amphipods☆ 
Data in Brief  2015;5:1-6.
Ovaries were taken from five sexually mature amphipods: Gammarus fossarum, Gammarus pulex, Gammarus roeseli, Hyallela azteca and Parhyale hawaiensis. The soluble proteome extracted from individual pair of ovaries from five biological replicates was trypsin digested and the resulting peptides were analyzed by high resolution tandem mass spectrometry. The spectra were assigned with four protein sequence databases with different specificities: a RNAseq-derived G. fossarum database; a RNAseq-derived P. hawaiensis database; both originating from ovaries transcriptome; the Daphnia pulex database derived from whole-genome sequencing and the NCBInr database. The best interpretation was obtained for most animals with the specific RNA-seq protein database previously established by means of RNAseq carried out on G. fossarum. Proteins identified in the five amphipod species allow defining the core-proteome of female reproductive tissues of the Senticaudata suborder. The data accompanying the manuscript describing the database searches and comparative analysis Trapp et al., 2015 [1] have been deposited to the ProteomeXchange with identifiers PXD002253 (G. fossarum), PXD002254 (G. pulex), PXD002255 (G. roeseli), PXD002256 (H. Azteca), and PXD002257 (P. hawaiensis).
doi:10.1016/j.dib.2015.07.037
PMCID: PMC4556749  PMID: 26380837
13.  Time dynamics of the Bacillus cereus exoproteome are shaped by cellular oxidation 
At low density, Bacillus cereus cells release a large variety of proteins into the extracellular medium when cultivated in pH-regulated, glucose-containing minimal medium, either in the presence or absence of oxygen. The majority of these exoproteins are putative virulence factors, including toxin-related proteins. Here, B. cereus exoproteome time courses were monitored by nanoLC-MS/MS under low-oxidoreduction potential (ORP) anaerobiosis, high-ORP anaerobiosis, and aerobiosis, with a specific focus on oxidative-induced post-translational modifications of methionine residues. Principal component analysis (PCA) of the exoproteome dynamics indicated that toxin-related proteins were the most representative of the exoproteome changes, both in terms of protein abundance and their methionine sulfoxide (Met(O)) content. PCA also revealed an interesting interconnection between toxin-, metabolism-, and oxidative stress–related proteins, suggesting that the abundance level of toxin-related proteins, and their Met(O) content in the B. cereus exoproteome, reflected the cellular oxidation under both aerobiosis and anaerobiosis.
doi:10.3389/fmicb.2015.00342
PMCID: PMC4406070  PMID: 25954265
exoproteome; Bacillus cereus; shotgun proteomics; methionine oxidation; toxins
14.  High-throughput, quantitative assessment of the effects of low-dose silica nanoparticles on lung cells: grasping complex toxicity with a great depth of field 
BMC Genomics  2015;16(1):315.
Background
The toxicity of manufactured fumed silica nanoparticles (NPs) remains poorly investigated compared to that of crystalline silica NPs, which have been associated with lung diseases after inhalation. Amorphous silica NPs are a raw material for manufactured nanocomposites, such as cosmetics, foods, and drugs, raising concerns about their potential toxicity.
Results
The size of the NPs was determined by dynamic light scattering and their shape was visualized by atomic force microscopy (10 ± 4 nm). The pertinent toxicological concentration and dynamic ranges were determined using viability tests and cellular impedance. We combined transcriptomics and proteomics to assess the cellular and molecular effects of fumed silica in A549 human alveolar epithelial cells. The “no observed transcriptomic adverse effect level” (NOTEL) was set to 1.0 μg/cm2, and the “lowest observed adverse transcriptional effect level” (LOTEL) was set at 1.5 μg/cm2. We carried out genome-wide expression profiles with microarrays and identified, by shotgun proteomics, the exoproteome changes in lung cells after exposure to NP doses (0.1, 1.0, 1.5, 3.0, and 6.0 μg/cm2) at two time points (24 h and 72 h). The data revealed a hierarchical, dose-dependent cellular response to silica NPs. At 1.5 μg/cm2, the Rho signaling cascade, actin cytoskeleton remodeling, and clathrin-mediated endocytosis were induced. At 3.0 μg/cm2, many inflammatory mediators were upregulated and the coagulation system pathway was triggered. Lastly, at 6.0 μg/cm2, oxidative stress was initiated. The proteins identified in the extracellular compartment were consistent with these findings.
Conclusions
The alliance of two high-throughput technologies allowed the quantitative assessment of the cellular effects and molecular consequences of exposure of lung cells to low doses of NPs. These results were obtained using a pathway-driven analysis instead of isolated genes. As in photography, toxicogenomics allows, at the same time, the visualization of a wide spectrum of biological responses and a “zoom in” to the details with a great depth of field. This study illustrates how such an approach based on human cell culture models is a valuable predictive screening tool to evaluate the toxicity of many potentially harmful emerging substances, alone or in mixtures, in the framework of future regulatory reinforcements.
Electronic supplementary material
The online version of this article (doi:10.1186/s12864-015-1521-5) contains supplementary material, which is available to authorized users.
doi:10.1186/s12864-015-1521-5
PMCID: PMC4404697  PMID: 25895662
Transcriptomics; Proteomics; Toxicogenomics; Nanoparticles; Silica; Cytotoxicity; Adverse outcome pathways; Cellular impedance
15.  Proteomic Evidences for Rex Regulation of Metabolism in Toxin-Producing Bacillus cereus ATCC 14579 
PLoS ONE  2014;9(9):e107354.
The facultative anaerobe, Bacillus cereus, causes diarrheal diseases in humans. Its ability to deal with oxygen availability is recognized to be critical for pathogenesis. The B. cereus genome comprises a gene encoding a protein with high similarities to the redox regulator, Rex, which is a central regulator of anaerobic metabolism in Bacillus subtilis and other Gram-positive bacteria. Here, we showed that B. cereus rex is monocistronic and down-regulated in the absence of oxygen. The protein encoded by rex is an authentic Rex transcriptional factor since its DNA binding activity depends on the NADH/NAD+ ratio. Rex deletion compromised the ability of B. cereus to cope with external oxidative stress under anaerobiosis while increasing B. cereus resistance against such stress under aerobiosis. The deletion of rex affects anaerobic fermentative and aerobic respiratory metabolism of B. cereus by decreasing and increasing, respectively, the carbon flux through the NADH-recycling lactate pathway. We compared both the cellular proteome and exoproteome of the wild-type and Δrex cells using a high throughput shotgun label-free quantitation approach and identified proteins that are under control of Rex-mediated regulation. Proteomics data have been deposited to the ProteomeXchange with identifier PXD000886. The data suggest that Rex regulates both the cross-talk between metabolic pathways that produce NADH and NADPH and toxinogenesis, especially in oxic conditions.
doi:10.1371/journal.pone.0107354
PMCID: PMC4162614  PMID: 25216269
16.  RNA Sequencing and Proteogenomics Reveal the Importance of Leaderless mRNAs in the Radiation-Tolerant Bacterium Deinococcus deserti 
Genome Biology and Evolution  2014;6(4):932-948.
Deinococcus deserti is a desiccation- and radiation-tolerant desert bacterium. Differential RNA sequencing (RNA-seq) was performed to explore the specificities of its transcriptome. Strikingly, for 1,174 (60%) mRNAs, the transcription start site was found exactly at (916 cases, 47%) or very close to the translation initiation codon AUG or GUG. Such proportion of leaderless mRNAs, which may resemble ancestral mRNAs, is unprecedented for a bacterial species. Proteomics showed that leaderless mRNAs are efficiently translated in D. deserti. Interestingly, we also found 173 additional transcripts with a 5′-AUG or 5′-GUG that would make them competent for ribosome binding and translation into novel small polypeptides. Fourteen of these are predicted to be leader peptides involved in transcription attenuation. Another 30 correlated with new gene predictions and/or showed conservation with annotated and nonannotated genes in other Deinococcus species, and five of these novel polypeptides were indeed detected by mass spectrometry. The data also allowed reannotation of the start codon position of 257 genes, including several DNA repair genes. Moreover, several novel highly radiation-induced genes were found, and their potential roles are discussed. On the basis of our RNA-seq and proteogenomics data, we propose that translation of many of the novel leaderless transcripts, which may have resulted from single-nucleotide changes and maintained by selective pressure, provides a new explanation for the generation of a cellular pool of small peptides important for protection of proteins against oxidation and thus for radiation/desiccation tolerance and adaptation to harsh environmental conditions.
doi:10.1093/gbe/evu069
PMCID: PMC4007540  PMID: 24723731
protein translation initiation; genome evolution; small peptides; desiccation tolerance; protein protection; transcription start sites
17.  Ribosomal proteins as biomarkers for bacterial identification by mass spectrometry in the clinical microbiology laboratory 
Whole-cell matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) is a rapid method for identification of microorganisms that is increasingly used in microbiology laboratories. This identification is based on the comparison of the tested isolate mass spectrum with reference databases. Using Neisseria meningitidis as a model organism, we showed that in one of the available databases, the Andromas database, 10 of the 13 species-specific biomarkers correspond to ribosomal proteins. Remarkably, one biomarker, ribosomal protein L32, was subject to inter-strain variability. The analysis of the ribosomal protein patterns of 100 isolates for which whole genome sequences were available, confirmed the presence of inter-strain variability in the molecular weight of 29 ribosomal proteins, thus establishing a correlation between the sequence type (ST) and/or clonal complex (CC) of each strain and its ribosomal protein pattern. Since the molecular weight of three of the variable ribosomal proteins (L30, L31 and L32) was included in the spectral window observed by MALDI-TOF MS in clinical microbiology, i.e., 3640–12000 m/z, we were able by analyzing the molecular weight of these three ribosomal proteins to classify each strain in one of six subgroups, each of these subgroups corresponding to specific STs and/or CCs. Their detection by MALDI-TOF allows therefore a quick typing of N. meningitidis isolates.
doi:10.1016/j.mimet.2013.07.021
PMCID: PMC3980635  PMID: 23916798
Mass spectrometry; Ribosomal proteins; Biomarkers; Neisseria meningitidis
18.  Low Doses of Gamma-Irradiation Induce an Early Bystander Effect in Zebrafish Cells Which Is Sufficient to Radioprotect Cells 
PLoS ONE  2014;9(3):e92974.
The term “bystander effect” is used to describe an effect in which cells that have not been exposed to radiation are affected by irradiated cells though various intracellular signaling mechanisms. In this study we analyzed the kinetics and mechanisms of bystander effect and radioadaptation in embryonic zebrafish cells (ZF4) exposed to chronic low dose of gamma rays. ZF4 cells were irradiated for 4 hours with total doses of gamma irradiation ranging from 0.01–0.1 Gy. In two experimental conditions, the transfer of irradiated cells or culture medium from irradiated cells results in the occurrence of DNA double strand breaks in non-irradiated cells (assessed by the number of γ-H2AX foci) that are repaired at 24 hours post-irradiation whatever the dose. At low total irradiation doses the bystander effect observed does not affect DNA repair mechanisms in targeted and bystander cells. An increase in global methylation of ZF4 cells was observed in irradiated cells and bystander cells compared to control cells. We observed that pre-irradiated cells which are then irradiated for a second time with the same doses contained significantly less γ-H2AX foci than in 24 h gamma-irradiated control cells. We also showed that bystander cells that have been in contact with the pre-irradiated cells and then irradiated alone present less γ-H2AX foci compared to the control cells. This radioadaptation effect is significantly more pronounced at the highest doses. To determine the factors involved in the early events of the bystander effect, we performed an extensive comparative proteomic study of the ZF4 secretomes upon irradiation. In the experimental conditions assayed here, we showed that the early events of bystander effect are probably not due to the secretion of specific proteins neither the oxidation of these secreted proteins. These results suggest that early bystander effect may be due probably to a combination of multiple factors.
doi:10.1371/journal.pone.0092974
PMCID: PMC3965492  PMID: 24667817
19.  Assessing the Exoproteome of Marine Bacteria, Lesson from a RTX-Toxin Abundantly Secreted by Phaeobacter Strain DSM 17395 
PLoS ONE  2014;9(2):e89691.
Bacteria from the Roseobacter clade are abundant in surface marine ecosystems as over 10% of bacterial cells in the open ocean and 20% in coastal waters belong to this group. In order to document how these marine bacteria interact with their environment, we analyzed the exoproteome of Phaeobacter strain DSM 17395. We grew the strain in marine medium, collected the exoproteome and catalogued its content with high-throughput nanoLC-MS/MS shotgun proteomics. The major component represented 60% of the total protein content but was refractory to either classical proteomic identification or proteogenomics. We de novo sequenced this abundant protein with high-resolution tandem mass spectra which turned out being the 53 kDa RTX-toxin ZP_02147451. It comprised a peptidase M10 serralysin domain. We explained its recalcitrance to trypsin proteolysis and proteomic identification by its unusual low number of basic residues. We found this is a conserved trait in RTX-toxins from Roseobacter strains which probably explains their persistence in the harsh conditions around bacteria. Comprehensive analysis of exoproteomes from environmental bacteria should take into account this proteolytic recalcitrance.
doi:10.1371/journal.pone.0089691
PMCID: PMC3933643  PMID: 24586966
20.  The Deinococcus radiodurans DR1245 Protein, a DdrB Partner Homologous to YbjN Proteins and Reminiscent of Type III Secretion System Chaperones 
PLoS ONE  2013;8(2):e56558.
The bacterium Deinococcus radiodurans exhibits an extreme resistance to ionizing radiation. A small subset of Deinococcus genus-specific genes were shown to be up-regulated upon exposure to ionizing radiation and to play a role in genome reconstitution. These genes include an SSB-like protein called DdrB. Here, we identified a novel protein encoded by the dr1245 gene as an interacting partner of DdrB. A strain devoid of the DR1245 protein is impaired in growth, exhibiting a generation time approximately threefold that of the wild type strain while radioresistance is not affected. We determined the three-dimensional structure of DR1245, revealing a relationship with type III secretion system chaperones and YbjN family proteins. Thus, DR1245 may display some chaperone activity towards DdrB and possibly other substrates.
doi:10.1371/journal.pone.0056558
PMCID: PMC3575483  PMID: 23441204
21.  Eukaryotic GPN-loop GTPases paralogs use a dimeric assembly reminiscent of archeal GPN 
Cell Cycle  2013;12(3):463-472.
GTPases are molecular switches that regulate a wide-range of cellular processes. The GPN-loop GTPase (GPN) is a sub-family of P-loop NTPase that evolved from a single gene copy in archaea to triplicate paralog genes in eukaryotes, each having a non-redundant essential function in cell. In Saccharomyces cerevisiae, yGPN1 and yGPN2 are involved in sister chromatid cohesion mechanism, whereas nothing is known regarding yGPN3 function. Previous high-throughput experiments suggested that GPN paralogs interaction may occur. In this work, GPN|GPN contact was analyzed in details using TAP-Tag approach, yeast two-hybrid assay, in silico energy computation and site-directed mutagenesis of a conserved Glu residue located at the center of the interaction interface. It is demonstrated that this residue is essential for cell viability. A chromatid cohesion assay revealed that, like yGPN1 and yGPN2, yGPN3 also plays a role in sister chromatid cohesion. These results suggest that all three GPN proteins act at the molecular level in sister chromatid cohesion mechanism as a GPN|GPN complex reminiscent of the homodimeric structure of PAB0955, an archaeal member of GPN-loop GTPase.
doi:10.4161/cc.23367
PMCID: PMC3587447  PMID: 23324351
GPN-loop-GTPase; chromatid cohesion; heterodimer; paralogous interactions; P-loop NTPase
22.  Major soluble proteome changes in Deinococcus deserti over the earliest stages following gamma-ray irradiation 
Proteome Science  2013;11:3.
Background
Deinococcus deserti VCD115 has been isolated from Sahara surface sand. This radiotolerant bacterium represents an experimental model of choice to understand adaptation to harsh conditions encountered in hot arid deserts. We analysed the soluble proteome dynamics in this environmentally relevant model after exposure to 3 kGy gamma radiation, a non-lethal dose that generates massive DNA damages. For this, cells were harvested at different time lapses after irradiation and their soluble proteome contents have been analysed by 2-DE and mass spectrometry.
Results
In the first stage of the time course we observed accumulation of DNA damage response protein DdrB (that shows the highest fold change ~11), SSB, and two different RecA proteins (RecAP and RecAC). Induction of DNA repair protein PprA, DNA damage response protein DdrD and the two gyrase subunits (GyrA and GyrB) was also detected. A response regulator of the SarP family, a type II site-specific deoxyribonuclease and a putative N-acetyltransferase are three new proteins found to be induced. In a more delayed stage, we observed accumulation of several proteins related to central metabolism and protein turn-over, as well as helicase UvrD and novel forms of both gyrase subunits differing in terms of isoelectric point and molecular weight.
Conclusions
Post-translational modifications of GyrA (N-terminal methionine removal and acetylation) have been evidenced and their significance discussed. We found that the Deide_02842 restriction enzyme, which is specifically found in D. deserti, is a new potential member of the radiation/desiccation response regulon, highlighting the specificities of D. deserti compared to the D. radiodurans model.
doi:10.1186/1477-5956-11-3
PMCID: PMC3564903  PMID: 23320389
Proteome; Post-translational modification; Irradiation; Early response; Reference 2D map; Kinetics; Hierarchical clustering
23.  Proteomic insights into the lifestyle of an environmentally relevant marine bacterium 
The ISME Journal  2011;6(1):124-135.
In terms of lifestyle, free-living bacteria are classified as either oligotrophic/specialist or opportunist/generalist. Heterogeneous marine environments such as coastal waters favour the establishment of marine generalist bacteria, which code for a large pool of functions. This is basically foreseen to cope with the heterogeneity of organic matter supplied to these systems. Nevertheless, it is not known what fraction of a generalist proteome is needed for house-keeping functions or what fraction is modified to cope with environmental changes. Here, we used high-throughput proteomics to define the proteome of Ruegeria pomeroyi DSS-3, a model marine generalist bacterium of the Roseobacter clade. We evaluated its genome expression under several natural environmental conditions, revealing the versatility of the bacterium to adapt to anthropogenic influence, poor nutrient concentrations or the presence of the natural microbial community. We also assayed 30 different laboratory incubations to increase proteome coverage and to dig further into the functional genomics of the bacterium. We established its core proteome and the proteome devoted to adaptation to general cellular physiological variations (almost 50%). We suggest that the other half of its theoretical proteome is the opportunist genetic pool devoted exclusively to very specific environmental conditions.
doi:10.1038/ismej.2011.86
PMCID: PMC3246242  PMID: 21776030
proteomics; core proteome; specialist; generalist; Roseobacters; functional genomics
24.  High-throughput proteogenomics of Ruegeria pomeroyi: seeding a better genomic annotation for the whole marine Roseobacter clade 
BMC Genomics  2012;13:73.
Background
The structural and functional annotation of genomes is now heavily based on data obtained using automated pipeline systems. The key for an accurate structural annotation consists of blending similarities between closely related genomes with biochemical evidence of the genome interpretation. In this work we applied high-throughput proteogenomics to Ruegeria pomeroyi, a member of the Roseobacter clade, an abundant group of marine bacteria, as a seed for the annotation of the whole clade.
Results
A large dataset of peptides from R. pomeroyi was obtained after searching over 1.1 million MS/MS spectra against a six-frame translated genome database. We identified 2006 polypeptides, of which thirty-four were encoded by open reading frames (ORFs) that had not previously been annotated. From the pool of 'one-hit-wonders', i.e. those ORFs specified by only one peptide detected by tandem mass spectrometry, we could confirm the probable existence of five additional new genes after proving that the corresponding RNAs were transcribed. We also identified the most-N-terminal peptide of 486 polypeptides, of which sixty-four had originally been wrongly annotated.
Conclusions
By extending these re-annotations to the other thirty-six Roseobacter isolates sequenced to date (twenty different genera), we propose the correction of the assigned start codons of 1082 homologous genes in the clade. In addition, we also report the presence of novel genes within operons encoding determinants of the important tricarboxylic acid cycle, a feature that seems to be characteristic of some Roseobacter genomes. The detection of their corresponding products in large amounts raises the question of their function. Their discoveries point to a possible theory for protein evolution that will rely on high expression of orphans in bacteria: their putative poor efficiency could be counterbalanced by a higher level of expression. Our proteogenomic analysis will increase the reliability of the future annotation of marine bacterial genomes.
doi:10.1186/1471-2164-13-73
PMCID: PMC3305630  PMID: 22336032
25.  Biosynthesis of Wyosine Derivatives in tRNA: An Ancient and Highly Diverse Pathway in Archaea 
Molecular Biology and Evolution  2010;27(9):2062-2077.
Wyosine (imG) and its derivatives such as wybutosine (yW) are found at position 37 of phenylalanine-specific transfer RNA (tRNAPhe), 3′ adjacent to the anticodon in Eucarya and Archaea. In Saccharomyces cerevisiae, formation of yW requires five enzymes acting in a strictly sequential order: Trm5, Tyw1, Tyw2, Tyw3, and Tyw4. Archaea contain wyosine derivatives, but their diversity is greater than in eukaryotes and the corresponding biosynthesis pathways still unknown. To identify these pathways, we analyzed the phylogenetic distribution of homologues of the yeast wybutosine biosynthesis proteins in 62 archaeal genomes and proposed a scenario for the origin and evolution of wyosine derivatives biosynthesis in Archaea that was partly experimentally validated. The key observations were 1) that four of the five wybutosine biosynthetic enzymes are ancient and may have been present in the last common ancestor of Archaea and Eucarya, 2) that the variations in the distribution pattern of biosynthesis enzymes reflect the diversity of the wyosine derivatives found in different Archaea. We also identified 7-aminocarboxypropyl-demethylwyosine (yW-86) and its N4-methyl derivative (yW-72) as final products in tRNAs of several Archaea when these were previously thought to be only intermediates of the eukaryotic pathway. We confirmed that isowyosine (imG2) and 7-methylwyosine (mimG) are two archaeal-specific guanosine-37 derivatives found in tRNA of both Euryarchaeota and Crenarchaeota. Finally, we proposed that the duplication of the trm5 gene in some Archaea led to a change in function from N1 methylation of guanosine to C7 methylation of 4-demethylwyosine (imG-14).
doi:10.1093/molbev/msq096
PMCID: PMC4481705  PMID: 20382657
tRNA; modification enzymes; methyltransferase; biosynthetic pathway; anticodon loop; mass spectrometry; phylogeny; evolution; Archaea

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