Insights into dynamics and gating properties of T2SS secretins.

Secretins are outer membrane (OM) channels found in various bacterial nanomachines that secrete or assemble large extracellular structures. High-resolution 3D structures of type 2 secretion system (T2SS) secretins revealed bimodular channels with a C-module, holding a conserved central gate and an optional top gate, followed by an N-module for which multiple structural organizations have been proposed. Here, we perform a structure-driven in vivo study of the XcpD secretin, which validates one of the organizations of the N-module whose flexibility enables alternative conformations. We also show the existence of the central gate in vivo and its required flexibility, which is key for substrate passage and watertightness control. Last, functional, genomic, and phylogenetic analyses indicate that the optional top gate provides a gain of watertightness. Our data illustrate how the gating properties of T2SS secretins allow these large channels to overcome the duality between the necessity of preserving the OM impermeability while simultaneously promoting the secretion of large, folded effectors.

Genomic evolution and adaptation of arthropod-associated Rickettsia.

Rickettsia species are endosymbionts hosted by arthropods and are known to cause mild to fatal diseases in humans. Here, we analyse the evolution and diversity of 34 Rickettsia species using a pangenomic meta-analysis (80 genomes/41 plasmids). Phylogenomic trees showed that Rickettsia spp. diverged into two Spotted Fever groups, a Typhus group, a Canadensis group and a Bellii group, and may have inherited their plasmids from an ancestral plasmid that persisted in some strains or may have been lost by others. The results suggested that the ancestors of Rickettsia spp. might have infected Acari and/or Insecta and probably diverged by persisting inside and/or switching hosts. Pangenomic analysis revealed that the Rickettsia genus evolved through a strong interplay between genome degradation/reduction and/or expansion leading to possible distinct adaptive trajectories. The genus mainly shared evolutionary relationships with α-proteobacteria, and also with γ/ß/δ-proteobacteria, cytophagia, actinobacteria, cyanobacteria, chlamydiia and viruses, suggesting lateral exchanges of several critical genes. These evolutionary processes have probably been orchestrated by an abundance of mobile genetic elements, especially in the Spotted Fever and Bellii groups. In this study, we provided a global evolutionary genomic view of the intracellular Rickettsia that may help our understanding of their diversity, adaptation and fitness.

New records of bacteria in different species of fleas from France and Spain.

In this study, we assessed the presence of vector-borne microorganisms in different species of fleas collected from different hosts in diverse areas of South-Western Europe by molecular methods. A total of 319 fleas belonging to eight different species was tested for the presence of eight microorganisms. Wolbachia spp. endosymbionts were detected in Ctenocephalides felis, Pulex irritans, Archaeopsylla erinacei and Ctenophthalmus baeticus boisseauorum specimens. Rickettsia felis, an emerging pathogen, was detected in C. felis, A. erinacei and Ct. b. boisseauorum. Rickettsia typhi, the agent of murine typhus was detected for the first time in A. erinacei and Mycobacterium spp. were detected for the first time in fleas (C. felis, P. irritans and A. erinacei). Lastly, five different species of Bartonella were detected in fleas' DNA in this study, including a possible new bacterium belonging to this genus. With this study, we updated the knowledge of the flea-borne bacteria present in the South-West of Europe reinforcing the idea about the necessity to expand and increase the current knowledge on flea-borne pathogens.

Genome sequence-based criteria for demarcation and definition of species in the genus Rickettsia.

Over recent years, genomic information has increasingly been used for prokaryotic species definition and classification. Genome sequence-based alternatives to the gold standard DNA-DNA hybridization (DDH) relatedness have been developed, notably average nucleotide identity (ANI), which is one of the most useful measurements for species delineation in the genomic era. However, the strictly intracellar lifestyle, the few measurable phenotypic properties and the low level of genetic heterogeneity made the current standard genomic criteria for bacterial species definition inapplicable to Rickettsia species. We evaluated a range of whole genome sequence (WGS)-based taxonomic parameters to develop guidelines for the classification of Rickettsia isolates at genus and species levels. By comparing the degree of similarity of 74 WGSs from 31 Rickettsia species and 61 WGSs from members of three closely related genera also belonging to the order Rickettsiales (Orientia, 11 genomes; Ehrlichia, 22 genomes; and Anaplasma, 28 genomes) using digital DDH (dDDh) and ANI by orthology (OrthoANI) parameters, we demonstrated that WGS-based taxonomic information, which is easy to obtain and use, can serve for reliable classification of isolates within the Rickettsia genus and species. To be classified as a member of the genus Rickettsia, a bacterial isolate should exhibit OrthoANI values with any Rickettsia species with a validly published name of ≥83.63 %. To be classified as a new Rickettsia species, an isolate should not exhibit more than any of the following degrees of genomic relatedness levels with the most closely related species: >92.30 and >99.19 % for the dDDH and OrthoANI values, respectively. When applied to four rickettsial isolates of uncertain status, the above-described thresholds enabled their classification as new species in one case. Thus, we propose WGS-based guidelines to efficiently delineate Rickettsia species, with OrthoANI and dDDH being the most accurate for classification at the genus and species levels, respectively.

Rapid MALDI-TOF MS identification of commercial truffles.

Truffles are edible mushrooms with similar morphological characteristics, that make it difficult to distinguish between highly prized truffles (such as the Périgord black T. melanosporum) and inexpensive truffles (such as the Asian Black T. indicum). These biological and economic features have led to several misidentifications and/or fraudulent profit in the truffle markets. In this paper, we investigate Matrix-assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS) biotyping to identify 34 commercial fresh truffles from Europe and Asia. The MALDI-TOF MS clustering rapidly distinguished seven Tuber species identified by ITS phylogenetic analysis. The tasty T. melanosporum was clearly differentiated from the Chinese and less expensive truffles. These cheaper mushrooms were marketed as T. indicum but corresponded to a mix of three species. In total, the method confirmed misidentifications in 26% of commercial specimens. Several unknown blind-coded truffles were rapidly identified, with scores >= 2, using the Bruker Biotyper algorithm against MS databases. This study demonstrates that MALDI-TOF MS is a reliable, rapid and cheaper new tool compared with molecular methods for the identification of truffle species and could be used to control frauds in the truffle markets. It could also be useful for the certification of truffle-inoculated seedlings and/or diversity in forest ecosystems.

Identification of rickettsial immunoreactive proteins using a proximity ligation assay Western blotting and the traditional immunoproteomic approach.

The closely related species Rickettsia conorii and R. africae are both etiological agents of rickettsiosis, a tick-borne serious infective disease. The laboratory diagnosis is based on serology, but remains not enough specific to provide the diagnosis at the species level. Here, we attempted to identify specific proteins that would enable the discrimination of R. africae sp from R. conorii sp infections. We screened 22 R. africae- and 24 R. conorii-infected sera at different course of infection using a traditional immunoproteomic approach. In parallel, we focused on the technical development of a "relatively new technique" named a proximity ligation assay coupled to two-dimensional Western blotting. The top range markers of R. africae early infection were rpoA, atpD, and acnA, ORF0029, R. africae active infection were rOmpB ß-peptide, OmpA, groEL and ORF1174, early R. conorii infection was prsA, RC0031, pepA, R. conorii active infection were ftsZ, cycM and rpoA. They are candidates for serodiagnosis of rickettsioses.

A gene transfer event suggests a long-term partnership between eustigmatophyte algae and a novel lineage of endosymbiotic bacteria.

Rickettsiales are obligate intracellular bacteria originally found in metazoans, but more recently recognized as widespread endosymbionts of various protists. One genus was detected also in several green algae, but reports on rickettsialean endosymbionts in other algal groups are lacking. Here we show that several distantly related eustigmatophytes (coccoid algae belonging to Ochrophyta, Stramenopiles) are infected by Candidatus Phycorickettsia gen. nov., a new member of the family Rickettsiaceae. The genome sequence of Ca. Phycorickettsia trachydisci sp. nov., an endosymbiont of Trachydiscus minutus CCALA 838, revealed genomic features (size, GC content, number of genes) typical for other Rickettsiales, but some unusual aspects of the gene content were noted. Specifically, Phycorickettsia lacks genes for several components of the respiration chain, haem biosynthesis pathway, or c-di-GMP-based signalling. On the other hand, it uniquely harbours a six-gene operon of enigmatic function that we recently reported from plastid genomes of two distantly related eustigmatophytes and from various non-rickettsialean bacteria. Strikingly, the eustigmatophyte operon is closely related to the one from Phycorickettsia, suggesting a gene transfer event between the endosymbiont and host lineages in early eustigmatophyte evolution. We hypothesize an important role of the operon in the physiology of Phycorickettsia infection and a long-term eustigmatophyte-Phycorickettsia coexistence.

Multi-omics Analysis Sheds Light on the Evolution and the Intracellular Lifestyle Strategies of Spotted Fever Group Rickettsia spp.

Arthropod-borne Rickettsia species are obligate intracellular bacteria which are pathogenic for humans. Within this genus, Rickettsia slovaca and Rickettsia conorii cause frequent and potentially severe infections, whereas Rickettsia raoultii and Rickettsia massiliae cause rare and milder infections. All four species belong to spotted fever group (SFG) rickettsiae. However, R. slovaca and R. raoultii cause scalp eschar and neck lymphadenopathy (SENLAT) and are mainly associated with Dermacentor ticks, whereas the other two species cause Mediterranean spotted fever (MSF) and are mainly transmitted by Rhipicephalus ticks. To identify the potential genes and protein profiles and to understand the evolutionary processes that could, comprehensively, relate to the differences in virulence and pathogenicity observed between these four species, we compared their genomes and proteomes. The virulent and milder agents displayed divergent phylogenomic evolution in two major clades, whereas either SENLAT or MSF disease suggests a discrete convergent evolution of one virulent and one milder agent, despite their distant genetic relatedness. Moreover, the two virulent species underwent strong reductive genomic evolution and protein structural variations, as well as a probable loss of plasmid(s), compared to the two milder species. However, an abundance of mobilome genes was observed only in the less pathogenic species. After infecting Xenopus laevis cells, the virulent agents displayed less up-regulated than down-regulated proteins, as well as less number of identified core proteins. Furthermore, their similar and distinct protein profiles did not contain some genes (e.g., ompA/B and rickA) known to be related to rickettsial adhesion, motility and/or virulence, but may include other putative virulence-, antivirulence-, and/or disease-related proteins. The identified evolutionary forces herein may have a strong impact on intracellular expressions and strategies in these rickettsiae, and that may contribute to the emergence of distinct virulence and diseases in humans. Thus, the current multi-omics data provide new insights into the evolution and fitness of SFG virulence and pathogenicity, and intracellular pathogenic bacteria.

Case Report: Scalp Eschar and Neck Lymphadenopathy Associated with Bacteremia due to Coxiella-Like Bacteria.

Coxiella-like bacteria have been recently proposed as human pathogens. Using molecular techniques, we detected Coxiella-like bacteria in the blood and serum samples of a patient with a scalp eschar, neck lymphadenopathy, severe urticaria, edema, fever, and arthralgia indicating that this organism can provide systemic complications.

Genome Sequence of the Tick-Borne Pathogen Rickettsia raoultii.

ITALIC! Rickettsia raoultiiis a tick-associated spotted fever group (SFG) organism, causing scalp eschar and neck lymphadenopathy after tick bite (SENLAT) in humans. We report here the genome sequence of ITALIC! R. raoultiistrain Khabarovsk(T)(CSUR R3(T), ATCC VR-1596(T)), which was isolated from a ITALIC! Dermacentor silvarumtick collected in Russia.

Origin and Evolution of Rickettsial Plasmids.

BACKGROUND: Rickettsia species are strictly intracellular bacteria that have undergone a reductive genomic evolution. Despite their allopatric lifestyle, almost half of the 26 currently validated Rickettsia species have plasmids. In order to study the origin, evolutionary history and putative roles of rickettsial plasmids, we investigated the evolutionary processes that have shaped 20 plasmids belonging to 11 species, using comparative genomics and phylogenetic analysis between rickettsial, microbial and non-microbial genomes. RESULTS: Plasmids were differentially present among Rickettsia species. The 11 species had 1 to 4 plasmid (s) with a size ranging from 12 kb to 83 kb. We reconstructed pRICO, the last common ancestor of the current rickettsial plasmids. pRICO was vertically inherited mainly from Rickettsia/Orientia chromosomes and diverged vertically into a single or multiple plasmid(s) in each species. These plasmids also underwent a reductive evolution by progressive gene loss, similar to that observed in rickettsial chromosomes, possibly leading to cryptic plasmids or complete plasmid loss. Moreover, rickettsial plasmids exhibited ORFans, recent gene duplications and evidence of horizontal gene transfer events with rickettsial and non-rickettsial genomes mainly from the α/γ-proteobacteria lineages. Genes related to maintenance and plasticity of plasmids, and to adaptation and resistance to stress mostly evolved under vertical and/or horizontal processes. Those involved in nucleotide/carbohydrate transport and metabolism were under the influence of vertical evolution only, whereas genes involved in cell wall/membrane/envelope biogenesis, cycle control, amino acid/lipid/coenzyme and secondary metabolites biosynthesis, transport and metabolism underwent mainly horizontal transfer events. CONCLUSION: Rickettsial plasmids had a complex evolution, starting with a vertical inheritance followed by a reductive evolution associated with increased complexity via horizontal gene transfer as well as gene duplication and genesis. The plasmids are plastic and mosaic structures that may play biological roles similar to or distinct from their co-residing chromosomes in an obligate intracellular lifestyle.

Genome Sequence of Rickettsia hoogstraalii, a Geographically Widely Distributed Tick-Associated Bacterium.

Rickettsia hoogstraalii is a tick-associated member of the spotted fever group rickettsiae that is geographically widely distributed. We report here the draft genome of R. hoogstraalii strain Croatica(T) (=DSM 22243 = UTMB 00003), which was isolated from Haemaphysalis sulcata ticks collected in Croatia.

Genome Sequence of Rickettsia tamurae, a Recently Detected Human Pathogen in Japan.

Rickettsia tamurae is a member of the spotted fever group rickettsiae, which was reported in 2011 to cause human infections in Japan. We report the draft genome sequence of R. tamurae strain AT-1(T), isolated from Amblyomma testudinarium ticks.

Draft Genome Sequence of Rickettsia aeschlimannii, Associated with Hyalomma marginatum Ticks.

Rickettsia aeschlimannii is a tick-associated human pathogen. We report here the draft genome of R. aeschlimannii strain MC16, isolated from Hyalomma marginatum marginatum ticks collected in Morocco.

Non contiguous-finished genome sequence and description of Enterobacter massiliensis sp. nov.

Enterobacter massiliensis strain JC163(T) sp. nov. is the type strain of E. massiliensis sp. nov., a new species within the genus Enterobacter. This strain, whose genome is described here, was isolated from the fecal flora of a healthy Senegalese patient. E. massiliensis is an aerobic rod. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 4,922,247 bp long genome (1 chromosome but no plasmid) exhibits a G+C content of 55.1% and contains 4,644 protein-coding and 80 RNA genes, including 5 rRNA genes.

Non-contiguous finished genome sequence and description of Bartonella senegalensis sp. nov.

Bartonella senegalensis sp. nov. strain OS02(T) is the type strain of B. senegalensis sp. nov., a new species within the genus Bartonella. This strain, whose genome is described here, was isolated in Senegal from the soft tick Ornithodoros sonrai, the vector of relapsing fever. B. senegalensis is an aerobic, rod-shaped, Gram-negative bacterium. Here we describe the features of this organism, together with the complete genome sequence and its annotation. The 1,966,996 bp-long genome contains 1,710 protein-coding and 46 RNA genes, including 6 rRNA genes.

Non-contiguous finished genome sequence and description of Bartonella florenciae sp. nov.

Bartonella florenciae sp. nov. strain R4(T) is the type strain of B. florenciae sp. nov., a new species within the genus Bartonella. This strain, whose genome is described here, was isolated in France from the spleen of the shrew Crocidura russula. B. florenciae is an aerobic, rod-shaped, Gram-negative bacterium. Here we describe the features of this organism, together with the complete genome sequence and its annotation. The 2,010,844 bp-long genome contains 1,909 protein-coding and 46 RNA genes, including two rRNA operons.

Genomic analysis of Rickettsia japonica strain YHT.

Rickettsia japonica strain YH, isolated in 1984 in Japan, is the type strain of R. japonica, the tick-borne agent of Japanese spotted fever. Here, we report the 1.33-Mb genome of this rickettsial species.

Genomic comparison of Kingella kingae strains.

Kingella kingae is a betaproteobacterium from the order Neisseriales, and it is an agent of invasive infections in children. We sequenced the genome from the septic arthritis strain 11220434. It is composed of a 1,990,794-bp chromosome but no plasmid, and it contains 2,042 protein-coding genes and 52 RNA genes, including 3 rRNA genes.

Genome sequence of Rickettsia conorii subsp. caspia, the agent of Astrakhan fever.

Rickettsia conorii subsp. caspia is the agent of Astrakhan fever, a spotted fever group rickettsiosis endemic to Astrakhan, Russia. The present study reports the draft genome of Rickettsia conorii subsp. caspia strain A-167.