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The phytopathogen Phytophthora parasitica, from the Oomycetes class, known to be the tobacco black shank agent, can induce devastating diseases in various crop, plant and forest ecosystems. The genus Phytophthora has been studied at the cellular level, suggesting that different developmental steps are induced by the expression of some specific genes. However, these studies have only been carried out on certain species, such as Phytophthora infestans and Phytophthora cactorum. As for Phytophthora parasitica, which can be considered as one of the top ten oomycete pathogens due to the economic impact and effect it has on food security, even less functional analyses and transcriptomics data are available. To date, little is known about the protein expression of Phytophthora parasitica, information that is essential for achieving a better understanding of this species. In this study, we aimed to gain insight into the proteomics of the mycelium of the Phytophthora parasitica strain INRA 310 by addressing the following questions: (i) how many predicted proteins can be detected on the mycelium of P. parasitica INRA 310, and (ii) what proteins can be detected? The proteomics experiments were performed on the mycelium of the strain Phytophthora parasitica INRA310, using the nanoliquid chromatography-MS/MS technique. A total of 219 proteins were identified, including ten unknown proteins and 209 proteins involved in lipid, carbohydrate, nucleotide, energy production and other metabolic pathways. This proteomics study is, to our knowledge, the first to be performed on the mycelium of Phytophthora parasitica INRA 310. It gives a brief first insight into its in vitro-expressed proteins. This work may be the first step before further, more comprehensive studies are undertaken with the aim of better understanding the biology of this species and its pathogenicity.

Here we report the discovery of Yaravirus, a lineage of amoebal virus with a puzzling origin and evolution. Yaravirus presents 80-nm-sized particles and a 44,924-bp dsDNA genome encoding for 74 predicted proteins. Yaravirus genome annotation showed that none of its genes matched with sequences of known organ-isms at the nucleotide level; at the amino acid level, six predicted proteins had distant matches in the nr database. Complimentary prediction of three-dimensional structures indicated possible func-tion of 17 proteins in total. Furthermore, we were not able to retrieve viral genomes closely related to Yaravirus in 8,535 publicly available metagenomes spanning diverse habitats around the globe. The Yaravirus genome also contained six types of tRNAs that did not match commonly used codons. Proteomics revealed that Yaravirus particles contain 26 viral proteins, one of which potentially represent-ing a divergent major capsid protein (MCP) with a predicted double jelly-roll domain. Structure-guided phylogeny of MCP suggests that Yaravirus groups together with the MCPs of Pleurochrysis endemic viruses. Yaravirus expands our knowledge of the diversity of DNA viruses. The phylogenetic distance between Yaravirus and all other viruses highlights our still preliminary assessment of the genomic diversity of eukaryotic viruses, reinforcing the need for the isolation of new viruses of protists.

The colibactin island (pks) of Escherichia coli formed by 19 genes (55-Kb), encodes non-ribosomal peptide (NRP) and polyketide (PK) synthases, which allow the synthesis of colibactin, a suspected hybrid PK-NRP compound that causes damage to DNA in eukaryotic cells. The clbP, an unusual essential gene, is found in the operon structure with the clbS gene in the pks-encoded machinery. Interestingly, the clbP gene has been annotated as a β-lactamase but no previous study has reported its β-lactamase characteristics. In this study, we (i) investigated the β-lactamase properties of the clbP gene in silico by analysing its phylogenetic relationship with bacterial β-lactamase and peptidase enzymes, (ii) compared its three-dimensional (3D) protein structure with those of bacterial β-lactamase proteins using the Phyr2 database and PyMOL software, and (iii) evaluated in vitro its putative enzymatic activities, including β-lactamase, nuclease, and ribonuclease using protein expression and purification from an E. coli BL21 strain. In this study, we reveal a structural configuration of toxin/antitoxin systems in this island. Thus, similar to the toxin/antitoxin systems, the role of the clbP gene within the pks-island gene group appears as an antitoxin, insofar as it is responsible for the activation of the toxin, which is colibactin. In silico, our analyses revealed that ClbP belonged to the superfamily of β-lactamase, class C. Furthermore, in vitro we were unable to demonstrate its β-lactamase activity, likely due to the fact that the clbP gene requires co-expression with other genes, such as the genes present in the pks-island (19 genes). More research is needed to better understand its actions, particularly with regards to antibiotics, and to discover whether it has any additional functions due to the importance of this gene and its toxicity.

Thienamycin, the first representative of carbapenem antibiotics was discovered in the mid-1970s from soil microorganism, Streptomyces cattleya, during the race to discover inhibitors of bacterial peptidoglycan synthesis. Chemically modified into imipenem (N-formimidoyl thienamycin), now one of the most clinically important antibiotics, thienamycin is encoded by a thienamycin gene cluster composed of 22 genes (thnA to thnV) from S. cattleya NRRL 8057 genome. Interestingly, the role of all thn-genes has been experimentally demonstrated in the thienamycin biosynthesis, except thnS, despite its annotation as putative β-lactamase. Here, we expressed thnS gene and investigated its activities against various substrates. Our analyses revealed that ThnS belonged to the superfamily of metallo-β-lactamase fold proteins. Compared to known β-lactamases such as OXA-48 and NDM-1, ThnS exhibited a lower affinity and less efficiency toward penicillin G and cefotaxime, while imipenem is more actively hydrolysed. Moreover, like most MBL fold enzymes, additional enzymatic activities of ThnS were detected such as hydrolysis of ascorbic acid, single strand DNA, and ribosomal RNA. ThnS appears as a MBL enzyme with multiple activities including a specialised β-lactamase activity toward imipenem. Thus, like toxin/antitoxin systems, the role of thnS gene within the thienamycin gene cluster appears as an antidote against the produced thienamycin.

Here we report the discovery of Yaravirus, a new lineage of amoebal virus with a puzzling origin and phylogeny. Yaravirus presents 80 nm-sized particles and a 44,924 bp dsDNA genome encoding for 74 predicted proteins. More than 90% (68) of Yaravirus predicted genes have never been described before, representing ORFans. Only six genes had distant homologs in public databases: an exonuclease/recombinase, a packaging-ATPase, a bifunctional DNA primase/polymerase and three hypothetical proteins. Furthermore, we were not able to retrieve viral genomes closely related to Yaravirus in 8,535 publicly available metagenomes spanning diverse habitats around the globe. The Yaravirus genome also contained six types of tRNAs that did not match commonly used codons. Proteomics revealed that Yaravirus particles contain 26 viral proteins, one of which potentially representing a novel capsid protein with no significant homology with NCLDV major capsid proteins but with a predicted double-jelly roll domain. Yaravirus expands our knowledge of the diversity of DNA viruses. The phylogenetic distance between Yaravirus and all other viruses highlights our still preliminary assessment of the genomic diversity of eukaryotic viruses, reinforcing the need for the isolation of new viruses of protists.Significance statementMost of the known viruses of amoeba have been seen to share many features that eventually prompted authors to classify them into common evolutionary groups. Here we describe Yaravirus, an entity that could represent either the first isolated virus of Acanthamoeba spp. out of the group of NCLDVs or, in alternative evolutive scenario, it is a distant and extremely reduced virus of this group. Contrary to what is observed in other isolated viruses of amoeba, Yaravirus is not represented by a large/giant particle and a complex genome, but at the same time carries an important number of previously undescribed genes, including one encoding a novel major capsid protein. Metagenomic approaches also testified for the rarity of Yaravirus in the environment.

Enzymatic proteins with a metallo-beta-lactamase (MBL) fold have been essentially studied in bacteria for their activity on beta-lactam antibiotics. However, the MBL fold is ancient and highly conserved, and these proteins are capable of cleaving a broad range of substrates. It has recently been shown that MBLs are present in a wide array of cellular organisms, including eukaryotes and archaea. We show here that Tupanvirus deep ocean, a giant virus, also encodes a protein with a MBL fold. Phylogeny showed its clustering with transfer ribonucleases (RNases) and the presence of orthologs in other giant viruses, mainly those harboring the largest sets of translation components. In addition, it suggests an ancient origin for these genes and a transfer between giant viruses and Acanthamoeba spp., a host of many giant viruses. Biologically, after its expression in Escherichia coli, the tupanvirus protein was found to hydrolyse nitrocefin, a chromogenic beta-lactam. We also observed an hydrolysis of penicillin G (10 μg/mL) and detected the metabolite of penicillin G hydrolysis, benzylpenilloic acid. This was inhibited by sulbactam, a beta-lactamase inhibitor. In addition, we tested the degradation of single-stranded DNA, double-stranded DNA, and RNAs, and observed a strong activity on RNAs from seven bacteria with G+C varying from 42% to 67%, and from Acanthamoeba castellanii, the tupanvirus host. This was not inhibited by sulbactam or ceftriaxone. RNase activity was estimated to be 0.45±0.15 mU/mg using a fluorescence-based assay. Our results still broaden the range of hosts of MBL fold proteins and demonstrate that such protein can have dual beta-lactamase/nuclease activities. We suggest that they should be annotated according to this finding to avoid further confusion.

β-lactams targeting the bacterial cell wall are not active on archaea. Here, we figure out that annotation of genes as β-lactamase in Archeae on the basis of homologous genes, initially annotated β-lactamases, is a remnant of the identification of the original activities of this group of enzymes, which in fact, have multiple functions including nuclease, ribonuclease, β-lactamase, or glyoxalase; which may specialized over time. We expressed a class B β-lactamase enzyme from Methanosarcina barkeri that digest penicillin G. Moreover, while a weak glyoxalase activity was detected, a significant ribonuclease activity on bacterial and synthetic RNAs was demonstrated. The β-lactamase activity was inhibited by a β-lactamase inhibitor (sulbactam), but its RNAse activity was not. This gene appears to has been transferred to the Flavobacteriaceae group including Elizabethkingia genus in which the expressed gene shows a more specialized activity toward resistance to tienanmicin but no glyoxalase activity. The expressed class C-like β-lactamase gene, also from Methanosarcina sp., shows also hydrolysis activity and was more closely related to DD-peptidase enzymes than known bacterial class C β-lactamases. Our findings highlight the requalification needness of annotated enzymes as β-lactamases and the specification overtime of multipotent enzymes in different ways in Archaea and bacteria.

The Acanthamoeba polyphaga mimivirus is the first giant virus ever described, with a 1.2-Mb genome which encodes 979 proteins, including central components of the translation apparatus. One of these proteins, R458, was predicted to initiate translation, although its specific role remains unknown. We silenced the R458 gene using small interfering RNA (siRNA) and compared levels of viral fitness and protein expression in silenced versus wild-type mimivirus. Silencing decreased the growth rate, but viral particle production at the end of the viral cycle was unaffected. A comparative proteomic approach using two-dimensional difference-in-gel electrophoresis (2D-DIGE) revealed deregulation of the expression of 32 proteins in silenced mimivirus, which were defined as up- or downregulated. Besides revealing proteins with unknown functions, silencing R458 also revealed deregulation in proteins associated with viral particle structures, transcriptional machinery, oxidative pathways, modification of proteins/lipids, and DNA topology/repair. Most of these proteins belong to genes transcribed at the end of the viral cycle. Overall, our data suggest that the R458 protein regulates the expression of mimivirus proteins and, thus, that mimivirus translational proteins may not be strictly redundant in relation to those from the amoeba host. As is the case for eukaryotic initiation factor 4a (eIF4a), the R458 protein is the prototypical member of the ATP-dependent DEAD box RNA helicase mechanism. We suggest that the R458 protein is required to unwind the secondary structures at the 5′ ends of mRNAs and to bind the mRNA to the ribosome, making it possible to scan for the start codon. These data are the first experimental evidence of mimivirus translation-related genes, predicted to initiate protein biosynthesis. IMPORTANCE The presence in the genome of a mimivirus of genes coding for many translational processes, with the exception of ribosome constituents, has been the subject of debate since its discovery in 2003. In this work, we focused on the R458 mimivirus gene, predicted to initiate protein biosynthesis. After silencing was performed, we observed that it has no major effect on mimivirus multiplication but that it affects protein expression and fitness. This suggests that it is effectively used by mimivirus during its developmental cycle. Until large-scale genetic manipulation of giant viruses becomes possible, the silencing strategy used here on mimivirus translation-related factors will open the way to understanding the functions of these translational genes.

BACKGROUND: Necrotizing enterocolitis (NEC) is the most common and serious gastrointestinal disorder among preterm neonates. We aimed to assess a specific gut microbiota profile associated with NEC. METHODS: Stool samples and clinical data were collected from 4 geographically independent neonatal intensive care units, over a 48-month period. Thirty stool samples from preterm neonates with NEC (n = 15) and controls (n = 15) were analyzed by 16S ribosomal RNA pyrosequencing and culture-based methods. The results led us to develop a specific quantitative polymerase chain reaction (qPCR) assay for Clostridium butyricum, and we tested stool samples from preterm neonates with NEC (n = 93) and controls (n = 270). We sequenced the whole genome of 16 C. butyricum strains, analyzed their phylogenetic relatedness, tested their culture supernatants for cytotoxic activity, and searched for secreted toxins. RESULTS: Clostridium butyricum was specifically associated with NEC using molecular and culture-based methods (15/15 vs 2/15; P < .0001) or qPCR (odds ratio, 45.4 [95% confidence interval, 26.2-78.6]; P < .0001). Culture supernatants of C. butyricum strains from preterm neonates with NEC (n = 14) exhibited significant cytotoxic activity (P = .008), and we identified in all a homologue of the β-hemolysin toxin gene shared by Brachyspira hyodysenteriae, the etiologic agent of swine dysentery. The corresponding protein was secreted by a NEC-associated C. butyricum strain. CONCLUSIONS: NEC was associated with C. butyricum strains and dysbiosis with an oxidized, acid, and poorly diversified gut microbiota. Our findings highlight the plausible toxigenic mechanism involved in the pathogenesis of NEC.

BACKGROUND Mimiviridae Mimivirus, including the largest known viruses, multiply in amoebae. Mimiviruses have been linked to pneumonia, but they have never been isolated from patients. To further understand the pathogenic role of these viruses, we aimed to isolate them from a patient presenting with pneumonia. METHODS We cultured, on Acanthamoeba polyphaga amoebae, pulmonary samples from 196 Tunisian patients with community-acquired pneumonia during the period 2009-2010. An improved technique was used for Mimivirus isolation, which used agar plates where the growth of giant viruses is revealed by the formation of lysis plaques. Mimivirus serology was tested by microimmunofluorescence and by bidimensional immunoproteomic analysis using Mimivirus strains, to identify specific immunoreactive proteins. The new Mimivirus strain genome sequencing was performed on Roche 454 GS FLX Titanium, then AB SOLiD instruments. RESULTS We successfully isolated a Mimivirus (LBA111), the largest virus ever isolated in a human sample, from a 72-year-old woman presenting with pneumonia. Electron microscopy revealed a Mimivirus-like virion with a size of 554 ± 10 nm. The LBA111 genome is 1.23 megabases, and it is closely related to that of Megavirus chilensis. Furthermore, the serum from the patient reacted specifically to the virus compared to controls. CONCLUSIONS This is the first Mimivirus isolated from a human specimen. The findings presented above together with previous works establish that mimiviruses can be associated with pneumonia. The common occurrence of these viruses in water and soil makes them probable global agents that are worthy of investigation.

Centre National de Re´fe´rence de la Re´sistance aux Antibiotiques, InstitutPasteur, Paris Cedex, FranceKempf M, Rolain J-M, Azza S, Diene S, Joly-Guillou M-L, Dubourg G, Colson P, Papazian L,Richet H, Fournier P-E, Ribeiro A, Raoult D. Investigation of Acinetobacter baumannii resistanceto carbapenems in Marseille hospitals, south of France: a transition from an epidemic to anendemic situation. APMIS 2012.Carbapenem-resistant Acinetobacter baumannii infections are a worldwide endemic nosocomialthreat. Between December 2010 and April 2011, an increase of carbapenem-resistant A. baumanniiinfections occurred in several Marseille University Hospitals. The aim of this study was to investi-gate the increase of carbapenem-resistant A. baumannii infections and to characterize the mecha-nisms of carbapenem resistance. The increase was detected by a homemade computer surveillanceprogram, known as EPIMIC, that monitors antibiotic resistance profiles on a weekly basis. Duringthis period, positive samples of carbapenem-resistant A. baumannii were retrieved from patients hos-pitalized in different units. Genotyping of the isolates was performed using pulsed-field gel electro-phoresis (PFGE) and multi-locus sequence typing (MLST), and carbapenemase gene analyses wereperformed to detect the presence of carbapenemases and to determine the relationships of theisolates. Carbapenem-resistant A. baumannii were isolated in a total of 11 patients who were hospi-talized in different hospitals units. We identified the presence of the bla

Bartonella henselae is an emerging gram-negative facultative intracellular pathogen transmitted via Ctenocephalides felis (cat fleas) or cat scratches. Bartonellosis is present mainly in the form of cat scratch disease (CSD), bacillary angiomatosis and infective endocarditis (IE). The methods used to diagnose B. henselae rely on culturing, immunofluorescent assays and molecular techniques. The objective of the present study was to identify candidate proteins for the serodiagnosis of bartonellosis with the differential discrimination of both clinical scenarios: CSD and IE. For this, an immunoproteomic approach combined with 2-DE, immunoblotting and matrix-assisted laser desorption/ionization time-of-flight MS has been developed. Immunoproteomic profiles of sera collected from patients with CSD and IE were compared with those of blood donors. We identified several candidate proteins as phage-encoding Pap31 protein and an outer membrane protein of BH11510 that, in our view, might be useful for the serodiagnosis of bartonellosis.

Whipple's disease (WD) is a chronic multisystemic infection, caused by the bacterium Tropheryma whipplei. The main clinical presentations are classic WD (CWD) with histologic lesions in the gastrointestinal tract, endocarditis, and isolated neurologic infection. The current strategy for diagnosis remains invasive.The present study aimed to select the protein candidates for serological diagnosis of WD. The first step was to identify candidate proteins by an immunoproteomic approach combining 2-DE using a total extract of a T. whipplei, immunoblotting, and MS. The second step was to validate the discovered biomarkers using a recombinant protein-based ELISA. Serum samples from 18 patients with WD and from 54 control individuals were tested. A sugar ABC transporter, TWT328 (sensitivity (Se) 61%, specificity (Sp) 87%, positive predictive value (PPV) 61%, negative predictive value (NPV) 87%, and positive likelihood ratio (PLR) 4.69) was the best marker for development of serodiagnosis for CWD. We also obtained a reproducible immunoreactive protein pattern for patients with isolated neurological infection due to T. whipplei (Se 100%, Sp 93%, PPV 55.5%, NPV 100%, and PLR 13.51) as an encouraging step towards noninvasive diagnosis of this particular manifestation. Nine recombinant candidates have been successfully screened with serum samples. Results from these ELISA assays skewed with those obtained with immunoblots.

Faustovirus, a new Asfarviridae-related giant virus, was recently isolated in Vermamoeba vermiformis, a protist found in sewage water in various geographical locations and occasionally reported in human eye infection cases. As part of a global metagenomic analysis of viral communities existing in biting midges, we report here for the first time the identification and isolation of a Faustovirus-like virus in hematophagous arthropods and its detection in their animal hosts. The DNA virome analysis of three pools of Culicoides sp., engorged female Culicoides imicola and non-engorged male/female C. imicola biting midges collected in Senegal, revealed the presence of amoeba-infecting giant viruses and, among them, a majority of sequences related to Faustovirus. Phylogenetic analyses conducted on several structural genes of Faustovirus confirmed the clustering of the arthropod-borne Faustovirus with sewage-borne Faustoviruses, with a distinct geographical clustering of Senegalese Faustovirus strains. Transmission electron microscopy identified viral particles with morphologies and diameters which were compatible with Faustovirus. The presence of infectious arthropod-borne Faustovirus was finally confirmed by successful isolation on V. vermiformis amoeba. Global proteomic analysis of biting midges identified that arthropods' blood meal originating from cattle, rodents and humans. Further screening of cattle sera and rodent tissue resulted in prevalence of Faustovirus being estimated at 38% in rodents and 14% in cattle, suggesting a possible origin of Faustovirus presence in arthropods via the ingestion of contaminated blood meal. Viral loads were the highest in rodents' urine and kidney samples, suggesting a possible excretion of viral particles into the environment. Faustovirus DNA polymerase-related sequences were also detected in more than 9 and 11% of febrile patients and healthy Senegalese human sera, respectively. Our study thus, highlights the need to investigate the role of arthropods, wildlife, and domestic animals in the lifecycle of amoeba-infecting giant viruses and, in particular, the environmental cycle of Faustovirus.

Background. Bacterial endocarditis is a serious disease. Surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectrometry (MS) based on serum protein profiling is a powerful approach that can generate biomarkers with diagnostic value. Methods. To identify a protein signature associated with bacterial endocarditis, we retrospectively performed SELDI-TOF MS profiling of serum samples from 88 patients hospitalized because of clinical suspicion of endocarditis. The diagnosis was confirmed by conventional criteria for 34 patients (endocarditis positive) and was excluded for 54 patients (endocarditis negative). Serum samples were incubated with cation-exchange ProteinChip arrays. The protein profiles generated were subjected to biostatistical processing. Results. Fifty-nine samples (23 endocarditis positive and 36 endocarditis negative) were randomly selected for a learning set, with the 29 remaining samples (11 endocarditis positive and 18 endocarditis negative) serving as an independent testing (validation) set. Sixty-six protein peaks were differentially expressed between the endocarditis-positive and the endocarditis-negative patients. By combining partial least squares and logistic regression methods, we built a serum protein model that perfectly discriminated between endocarditis-positive and endocarditis-negative patients. Importantly, when this model was tested on the independent testing set, a correct prediction rate of nearly 90% was demonstrated. Overall, sensitivity, specificity, positive predictive value, and negative predictive value were 94%, 98%, 96%, and 96%, respectively. Conclusions. SELDI-TOF MS profiling revealed a serum signature with high diagnostic potential for endocarditis.

The aldehyde dehydrogenases (ALDHs) are a superfamily of multimeric enzymes which catalyse the oxidation of a broad range of aldehydes into their corresponding carboxylic acids with the reduction of their cofactor, NAD or NADP, into NADH or NADPH. At present, the only known structures concern NAD-dependent ALDHs. Three structures are available in the Protein Data Bank: two are tetrameric and the other is a dimer. We solved by molecular replacement the first structure of an NADP-dependent ALDH isolated from Streptococcus mutans, in its apo form and holo form in complex with NADP, at 1.8 and 2.6 A resolution, respectively. Although the protein sequence shares only approximately 30 % identity with the other solved tetrameric ALDHs, the structures are very similar. However, a large local conformational change in the region surrounding the 2′ phosphate group of the adenosine moiety is observed when the enzyme binds NADP, in contrast to the NAD-dependent ALDHs. Structure and sequence analyses reveal several properties. A small number of residues seem to determine the oligomeric state. Likewise, the nature (charge and volume) of the residue at position 180 (Thr in ALDH from S. mutans) determines the cofactor specificity in comparison with the structures of NAD-dependent ALDHs. The presence of a hydrogen bond network around the cofactor not only allows it to bind to the enzyme but also directs the side-chains in a correct orientation for the catalytic reaction to take place. Moreover, a specific part of this network appears to be important in substrate binding. Since the enzyme oxidises the same substrate, glyceraldehyde-3-phosphate (G3P), as NAD-dependent phosphorylating glyceraldehyde-3-phosphate dehydrogenases (GAPDH), the active site of GAPDH was compared with that of the S. mutans ALDH. It was found that Arg103, Arg283 and Asp440 might be key residues for substrate binding.

GapB-encoded protein of Escherichia coli and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) share more than 40% amino acid identity. Most of the amino acids involved in the binding of cofactor and substrates to GAPDH are conserved in GapB-encoded protein. This enzyme shows an efficient non-phosphorylating erythrose-4-phosphate dehydrogenase activity (Zhao, G., Pease, A. J., Bharani, N., and Winkler, M. E. (1995) J. Bacteriol. 177, 2804-2812) but a low phosphorylating glyceraldehyde-3-phosphate dehydrogenase activity, whereas GAPDH shows a high efficient phosphorylating glyceraldehyde-3-phosphate dehydrogenase activity and a low phosphorylating erythrose-4-phosphate dehydrogenase activity. To identify the structural factors responsible for these differences, comparative kinetic and binding studies have been carried out on both GapB-encoded protein of Escherichia coli and GAPDH of Bacillus stearothermophilus. The KD constant of GapB-encoded protein for NAD is 800-fold higher than that of GAPDH. The chemical mechanism of erythrose 4-phosphate oxidation by GapB-encoded protein is shown to proceed through a two-step mechanism involving covalent intermediates with Cys-149, with rates associated to the acylation and deacylation processes of 280 s-1 and 20 s-1, respectively. No isotopic solvent effect is observed suggesting that the rate-limiting step is not hydrolysis. The rate of oxidation of glyceraldehyde 3-phosphate is 0.12 s-1 and is hydride transfer limiting, at least 2000-fold less efficient compared with that of erythrose 4-phosphate. Thus, it can be concluded that it is only the structure of the substrates that prevails in forming a ternary complex enzyme-NAD-thiohemiacetal productive (or not) for hydride transfer in the acylation step. This conclusion is reinforced by the fact that the rate of oxidation for erythrose 4-phosphate by GAPDH is 0.1 s-1 and is limited by the acylation step, whereas glyceraldehyde 3-phosphate acylation is efficient and is not rate-determining (/=800 s-1). Substituting Asn for His-176 on GapB-encoded protein, a residue postulated to facilitate hydride transfer as a base catalyst, decreases 40-fold the kcat of glyceraldehyde 3-phosphate oxidation. This suggests that the non-efficient positioning of the C-1 atom of glyceraldehyde 3-phosphate relative to the pyridinium of the cofactor within the ternary complex is responsible for the low catalytic efficiency. No phosphorylating activity on erythrose 4-phosphate with GapB-encoded protein is observed although the Pi site is operative as proven by the oxidative phosphorylation of glyceraldehyde 3-phosphate. Thus the binding of inorganic phosphate to the Pi site likely is not productive for attacking efficiently the thioacyl intermediate formed with erythrose 4-phosphate, whereas a water molecule is an efficient nucleophile for the hydrolysis of the thioacyl intermediate. Compared with glyceraldehyde-3-phosphate dehydrogenase activity, this corresponds to an activation of the deacylation step by/=4.5 kcal.mol-1. Altogether these results suggest subtle structural differences between the active sites of GAPDH and GapB-encoded protein that could be revealed and/or modulated by the structure of the substrate bound. This also indicates that a protein engineering approach could be used to convert a phosphorylating aldehyde dehydrogenase into an efficient non-phosphorylating one and vice versa.

International audience; Here, we sequenced the 5,419,609 bp circular genome of an Enterobacter aerogenes clinical isolate that killed a patient and was resistant to almost all current antibiotics (except gentamicin) commonly used to treat Enterobacterial infections, including colistin. Genomic and phylogenetic analyses explain the discrepancies of this bacterium and show that its core genome originates from another genus, Klebsiella. Atypical characteristics of this bacterium (i.e., motility, presence of ornithine decarboxylase, and lack of urease activity) are attributed to genomic mosaicism, by acquisition of additional genes, such as the complete 60,582 bp flagellar assembly operon acquired "en bloc" from the genus Serratia. The genealogic tree of the 162,202 bp multidrug-resistant conjugative plasmid shows that it is a chimera of transposons and integrative conjugative elements from various bacterial origins, resembling a rhizome. Moreover, we demonstrate biologically that a G53S mutation in the pmrA gene results in colistin resistance. E. aerogenes has a large RNA population comprising 8 rRNA operons and 87 cognate tRNAs that have the ability to translate transferred genes that use different codons, as exemplified by the significantly different codon usage between genes from the core genome and the "mobilome." On the basis of our findings, the evolution of this bacterium to become a "killer bug" with new genomic repertoires was from three criteria that are "opportunity, power, and usage" to indicate a sympatric lifestyle: "opportunity" to meet other bacteria and exchange foreign sequences since this bacteria was similar to sympatric bacteria; "power" to integrate these foreign sequences such as the acquisition of several mobile genetic elements (plasmids, integrative conjugative element, prophages, transposons, flagellar assembly system, etc.) found in his genome; and "usage" to have the ability to translate these sequences including those from rare codons to serve as a translator of foreign languages.

Most phagocytic protist viruses have large particles and genomes as well as many laterally acquired genes that may be associated with a sympatric intracellular life (a community-associated lifestyle with viruses, bacteria, and eukaryotes) and the presence of virophages. By subculturing Mimivirus 150 times in a germ-free amoebal host, we observed the emergence of a bald form of the virus that lacked surface fibers and replicated in a morphologically different type of viral factory. When studying a 0.40-μm filtered cloned particle, we found that its genome size shifted from 1.2 (M1) to 0.993 Mb (M4), mainly due to large deletions occurring at both ends of the genome. Some of the lost genes are encoding enzymes required for posttranslational modification of the structural viral proteins, such as glycosyltransferases and ankyrin repeat proteins. Proteomic analysis allowed identification of three proteins, probably required for the assembly of virus fibers. The genes for two of these were found to be deleted from the M4 virus genome. The proteins associated with fibers are highly antigenic and can be recognized by mouse and human antimimivirus antibodies. In addition, the bald strain (M4) was not able to propagate the sputnik virophage. Overall, the Mimivirus transition from a sympatric to an allopatric lifestyle was associated with a stepwise genome reduction and the production of a predominantly bald virophage resistant strain. The new axenic ecosystem allowed the allopatric Mimivirus to lose unnecessary genes that might be involved in the control of competitors.

Bartonella henselae is an emerging gram-negative facultative intracellular pathogen transmitted via Ctenocephalides felis (cat fleas) or cat scratches. Bartonellosis is present mainly in the form of cat scratch disease (CSD), bacillary angiomatosis and infective endocarditis (IE). The methods used to diagnose B. henselae rely on culturing, immunofluorescent assays and molecular techniques. The objective of the present study was to identify candidate proteins for the serodiagnosis of bartonellosis with the differential discrimination of both clinical scenarios: CSD and IE. For this, an immunoproteomic approach combined with 2-DE, immunoblotting and matrix-assisted laser desorption/ionization time-of-flight MS has been developed. Immunoproteomic profiles of sera collected from patients with CSD and IE were compared with those of blood donors. We identified several candidate proteins as phage-encoding Pap31 protein and an outer membrane protein of BH11510 that, in our view, might be useful for the serodiagnosis of bartonellosis.

Giant viruses such as Mimivirus isolated from amoeba found in aquatic habitats show biological sophistication comparable to that of simple cellular life forms and seem to evolve by similar mechanisms, including extensive gene duplication and horizontal gene transfer (HGT), possibly in part through a viral parasite, the virophage. We report here the isolation of “Marseille” virus, a previously uncharacterized giant virus of amoeba. The virions of Marseillevirus encompass a 368-kb genome, a minimum of 49 proteins, and some messenger RNAs. Phylogenetic analysis of core genes indicates that Marseillevirus is the prototype of a family of nucleocytoplasmic large DNA viruses (NCLDV) of eukaryotes. The genome repertoire of the virus is composed of typical NCLDV core genes and genes apparently obtained from eukaryotic hosts and their parasites or symbionts, both bacterial and viral. We propose that amoebae are “melting pots” of microbial evolution where diverse forms emerge, including giant viruses with complex gene repertoires of various origins.

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.

International audience; In this work, we present a comparative two-dimensional (2D) PAGE analysis of Rickettsia conorii and Rickettsia prowazekii. This analysis reveals protein spots that were either unique to or common to both strains, some of them being identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry.

The adipamidase of a mutant strain of Brevibacterium sp. involved in the degradation of adiponitrile into adipic acid was purified and characterized. The molecular wight of the native enzyme was estimated at 120,000 and that of the sub-units at 58,000, suggesting that this is a dimer structure. This enzyme has a large activity spectrum; all the amides tested were found to be substrates of the adipamidase. An acyltransferase activity was also detected for short chain substrates (acetamide, acetic acid), as well as long chain substrates (oleic acid).

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A distinct class of infectious agents, the virophages that infect giant viruses of the Mimiviridae family, has been recently described. Here we report the simultaneous discovery of a giant virus of Acanthamoeba polyphaga (Lentille virus) that contains an integrated genome of a virophage (Sputnik 2), and a member of a previously unknown class of mobile genetic elements, the transpovirons. The transpovirons are linear DNA elements of ∼7 kb that encompass six to eight protein-coding genes, two of which are homologous to virophage genes. Fluorescence in situ hybridization showed that the free form of the transpoviron replicates within the giant virus factory and accumulates in high copy numbers inside giant virus particles, Sputnik 2 particles, and amoeba cytoplasm. Analysis of deep-sequencing data showed that the virophage and the transpoviron can integrate in nearly any place in the chromosome of the giant virus host and that, although less frequently, the transpoviron can also be linked to the virophage chromosome. In addition, integrated fragments of transpoviron DNA were detected in several giant virus and Sputnik genomes. Analysis of 19 Mimivirus strains revealed three distinct transpovirons associated with three subgroups of Mimiviruses. The virophage, the transpoviron, and the previously identified self-splicing introns and inteins constitute the complex, interconnected mobilome of the giant viruses and are likely to substantially contribute to interviral gene transfer.