Multiple Origins and Specific Evolution of CRISPR/Cas9 Systems in Minimal Bacteria (Mollicutes).

CRISPR/Cas systems provide adaptive defense mechanisms against invading nucleic acids in prokaryotes. Because of its interest as a genetic tool, the Type II CRISPR/Cas9 system from Streptococcus pyogenes has been extensively studied. It includes the Cas9 endonuclease that is dependent on a dual-guide RNA made of a tracrRNA and a crRNA. Target recognition relies on crRNA annealing and the presence of a protospacer adjacent motif (PAM). Mollicutes are currently the bacteria with the smallest genome in which CRISPR/Cas systems have been reported. Many of them are pathogenic to humans and animals (mycoplasmas and ureaplasmas) or plants (phytoplasmas and some spiroplasmas). A global survey was conducted to identify and compare CRISPR/Cas systems found in the genome of these minimal bacteria. Complete or degraded systems classified as Type II-A and less frequently as Type II-C were found in the genome of 21 out of 52 representative mollicutes species. Phylogenetic reconstructions predicted a common origin of all CRISPR/Cas systems of mycoplasmas and at least two origins were suggested for spiroplasmas systems. Cas9 in mollicutes were structurally related to the S. aureus Cas9 except the PI domain involved in the interaction with the PAM, suggesting various PAM might be recognized by Cas9 of different mollicutes. Structure of the predicted crRNA/tracrRNA hybrids was conserved and showed typical stem-loop structures pairing the Direct Repeat part of crRNAs with the 5' region of tracrRNAs. Most mollicutes crRNA/tracrRNAs showed G + C% significantly higher than the genome, suggesting a selective pressure for maintaining stability of these secondary structures. Examples of CRISPR spacers matching with mollicutes phages were found, including the textbook case of Mycoplasma cynos strain C142 having no prophage sequence but a CRISPR/Cas system with spacers targeting prophage sequences that were found in the genome of another M. cynos strain that is devoid of a CRISPR system. Despite their small genome size, mollicutes have maintained protective means against invading DNAs, including restriction/modification and CRISPR/Cas systems. The apparent lack of CRISPR/Cas systems in several groups of species including main pathogens of humans, ruminants, and plants suggests different evolutionary routes or a lower risk of phage infection in specific ecological niches.

Large-Scale Measurement of mRNA Degradation in Escherichia coli: To Delay or Not to Delay.

In this study, we compared different computational methods used for genome-wide determination of mRNA half-lives in Escherichia coli with a special focus on the impact on considering a delay before the onset of mRNA decay after transcription arrest. A wide variety of datasets were analyzed coming from different technical methods for mRNA quantification (microarrays, RNA-seq, and RT-qPCR) and different bacterial growth conditions. The exponential decay of mRNA levels was fitted using both linear and exponential models and with or without a delay. We showed that for all the models, independently of mRNA quantification methods and growth conditions, ignoring the delay resulted in only a modest overestimation of the half-life. For approximately 80% of the mRNAs, differences in mRNA half-life values were less than 34s. The correlation between half-lives estimated with and without a delay was extremely high. However, the slope of the linear regression between the half-lives with and without a delay tended to decrease with the delay. For the few mRNAs for which taking into account the delay influenced the estimated half-life, the impact was dependent on the model and the growth condition. The smallest impact was obtained for the linear model.

Unraveling the evolution and coevolution of small regulatory RNAs and coding genes in Listeria.

BACKGROUND: Small regulatory RNAs (sRNAs) are widely found in bacteria and play key roles in many important physiological and adaptation processes. Studying their evolution and screening for events of coevolution with other genomic features is a powerful way to better understand their origin and assess a common functional or adaptive relationship between them. However, evolution and coevolution of sRNAs with coding genes have been sparsely investigated in bacterial pathogens. RESULTS: We designed a robust and generic phylogenomics approach that detects correlated evolution between sRNAs and protein-coding genes using their observed and inferred patterns of presence-absence in a set of annotated genomes. We applied this approach on 79 complete genomes of the Listeria genus and identified fifty-two accessory sRNAs, of which most were present in the Listeria common ancestor and lost during Listeria evolution. We detected significant coevolution between 23 sRNA and 52 coding genes and inferred the Listeria sRNA-coding genes coevolution network. We characterized a main hub of 12 sRNAs that coevolved with genes encoding cell wall proteins and virulence factors. Among them, an sRNA specific to L. monocytogenes species, rli133, coevolved with genes involved either in pathogenicity or in interaction with host cells, possibly acting as a direct negative post-transcriptional regulation. CONCLUSIONS: Our approach allowed the identification of candidate sRNAs potentially involved in pathogenicity and host interaction, consistent with recent findings on known pathogenicity actors. We highlight four sRNAs coevolving with seven internalin genes, some of which being important virulence factors in Listeria.

Genome-wide investigation of mRNA lifetime determinants in Escherichia coli cells cultured at different growth rates.

BACKGROUND: Changes to mRNA lifetime adjust mRNA concentration, facilitating the adaptation of growth rate to changes in growth conditions. However, the mechanisms regulating mRNA lifetime are poorly understood at the genome-wide scale and have not been investigated in bacteria growing at different rates. RESULTS: We used linear covariance models and the best model selected according to the Akaike information criterion to identify and rank intrinsic and extrinsic general transcript parameters correlated with mRNA lifetime, using mRNA half-life datasets for E. coli, obtained at four growth rates. The principal parameter correlated with mRNA stability was mRNA concentration, the mRNAs most concentrated in the cells being the least stable. However, sequence-related features (codon adaptation index (CAI), ORF length, GC content, polycistronic mRNA), gene function and essentiality also affected mRNA lifetime at all growth rates. We also identified sequence motifs within the 5'UTRs potentially related to mRNA stability. Growth rate-dependent effects were confined to particular functional categories (e.g. carbohydrate and nucleotide metabolism). Finally, mRNA stability was less strongly correlated with the amount of protein produced than mRNA concentration and CAI. CONCLUSIONS: This study provides the most complete genome-wide analysis to date of the general factors correlated with mRNA lifetime in E. coli. We have generalized for the entire population of transcripts or excluded determinants previously defined as regulators of stability for some particular mRNAs and identified new, unexpected general indicators. These results will pave the way for discussions of the underlying mechanisms and their interaction with the growth physiology of bacteria.

Diversity of CRISPR systems in the euryarchaeal Pyrococcales.

Pyrococcales are members of the order Thermococcales, a group of hyperthermophilic euryarchaea that are frequently found in deep sea hydrothermal vents. Infectious genetic elements, such as plasmids and viruses, remain a threat even in this remote environment and these microorganisms have developed several ways to fight their genetic invaders. Among these are the recently discovered CRISPR systems. In this review, we have combined and condensed available information on genetic elements infecting the Thermococcales and on the multiple CRISPR systems found in the Pyrococcales to fight them. Their organization and mode of action will be presented with emphasis on the Type III-B system that is the only CRISPR system known to target RNA molecules in a process reminiscent of RNA interference. The intriguing case of Pyrococcus abyssi, which is among the rare strains to present a CRISPR system devoid of the universal cas1 and cas2 genes, is also discussed.

Identification of CRISPR and riboswitch related RNAs among novel noncoding RNAs of the euryarchaeon Pyrococcus abyssi.

BACKGROUND: Noncoding RNA (ncRNA) has been recognized as an important regulator of gene expression networks in Bacteria and Eucaryota. Little is known about ncRNA in thermococcal archaea except for the eukaryotic-like C/D and H/ACA modification guide RNAs. RESULTS: Using a combination of in silico and experimental approaches, we identified and characterized novel P. abyssi ncRNAs transcribed from 12 intergenic regions, ten of which are conserved throughout the Thermococcales. Several of them accumulate in the late-exponential phase of growth. Analysis of the genomic context and sequence conservation amongst related thermococcal species revealed two novel P. abyssi ncRNA families. The CRISPR family is comprised of crRNAs expressed from two of the four P. abyssi CRISPR cassettes. The 5'UTR derived family includes four conserved ncRNAs, two of which have features similar to known bacterial riboswitches. Several of the novel ncRNAs have sequence similarities to orphan OrfB transposase elements. Based on RNA secondary structure predictions and experimental results, we show that three of the twelve ncRNAs include Kink-turn RNA motifs, arguing for a biological role of these ncRNAs in the cell. Furthermore, our results show that several of the ncRNAs are subjected to processing events by enzymes that remain to be identified and characterized. CONCLUSIONS: This work proposes a revised annotation of CRISPR loci in P. abyssi and expands our knowledge of ncRNAs in the Thermococcales, thus providing a starting point for studies needed to elucidate their biological function.

Biodistribution of radiolabelled human dendritic cells injected by various routes.

PURPOSE: The purpose of this study was to investigate the biodistribution of mature dendritic cells (DCs) injected by various routes, during a cell therapy protocol. METHODS: In the context of a vaccine therapy protocol for melanoma, DCs matured with Ribomunyl and interferon-gamma were labelled with( 111)In-oxine and injected into eight patients along various routes: afferent lymphatic vessel (IL) (4 times), lymph node (IN) (5 times) and intradermally (ID) (6 times). RESULTS: Scintigraphic investigations showed that the IL route allowed localisation of 80% of injected radioactivity in eight to ten nodes. In three cases of IN injection, the entire radioactivity stagnated in the injected nodes, while in two cases, migration to adjacent nodes was observed. This migration was detected rapidly after injection, as with IL injections, suggesting that passive transport occurred along the physiological lymphatic pathways. In two of the six ID injections, 1-2% of injected radioactivity reached a proximal lymph node. Migration was detectable in the first hour, but increased considerably after 24 h, suggesting an active migration mechanism. In both of the aforementioned cases, DCs were strongly CCR7-positive, but this feature was not a sufficient condition for effective migration. In comparison with DCs matured with TNF-alpha, IL-1beta, IL-6 and PGE2, our DCs showed a weaker in vitro migratory response to CCL21, despite comparable CCR7 expression, and higher allostimulatory and TH1 polarisation capacities. CONCLUSION: The IL route allowed reproducible administration of specified numbers of DCs. The IN route sometimes yielded fairly similar results, but not reproducibly. Lastly, we showed that DCs matured without PGE2 that have in vitro TH1 polarisation capacities can migrate to lymph nodes after ID injection.

Development of 99mTc labelled Lipiodol: biodistribution following injection into the hepatic artery of the healthy pig.

BACKGROUND: We develop a method for the radiolabelling of Lipiodol with Tc, using a lipophilic complex, [99mTc-(S2CPh)(S3Ph)2], dissolved in Lipiodol (99mTc-SSS Lipiodol). RESULTS: The labelling yield is high (96 +/- 0.8%), and the radiochemical purity satisfactory (92 +/- 2.6%). This labelling is reproducible and stable for up to 24 h in vitro. Studies carried out after injection into the hepatic artery of the healthy pig show that the biodistribution of 99mTc-SSS Lipiodol is comparable with that observed for 188Re Lipiodol. MATERIALS AND METHODS: The 99mTc-SSS lipiodol was obtained after dissolving a chelating agent, previously labelled with 99mTc, in cold lipiodol. The radiochemical purity (RCP) of the labelling was checked immediately and at 24 h. The 99mTc-SSS lipiodol was injected into the hepatic artery of four healthy pigs for an ex-vivo biodistribution study. An autoradiographic study was performed in two cases. CONCLUSIONS: Apart from the specific interest of a Lipiodol-bearing technetiated agent for carrying out dosimetric studies, the labelling of Lipiodol with 99mTc is a preliminary step towards the use of radiolabelling with the 188Re analogue.

FrameD: A flexible program for quality check and gene prediction in prokaryotic genomes and noisy matured eukaryotic sequences.

We describe FrameD, a program that predicts coding regions in prokaryotic and matured eukaryotic sequences. Initially targeted at gene prediction in bacterial GC rich genomes, the gene model used in FrameD also allows to predict genes in the presence of frameshifts and partially undetermined sequences which makes it also very suitable for gene prediction and frameshift correction in unfinished sequences such as EST and EST cluster sequences. Like recent eukaryotic gene prediction programs, FrameD also includes the ability to take into account protein similarity information both in its prediction and its graphical output. Its performances are evaluated on different bacterial genomes. The web site (http://genopole.toulouse.inra.fr/bioinfo/FrameD/FD) allows direct prediction, sequence correction and translation and the ability to learn new models for new organisms.

EUGENE'HOM: A generic similarity-based gene finder using multiple homologous sequences.

EUGENE'HOM is a gene prediction software for eukaryotic organisms based on comparative analysis. EUGENE'HOM is able to take into account multiple homologous sequences from more or less closely related organisms. It integrates the results of TBLASTX analysis, splice site and start codon prediction and a robust coding/non-coding probabilistic model which allows EUGENE'HOM to handle sequences from a variety of organisms. The current target of EUGENE'HOM is plant sequences. The EUGENE'HOM web site is available at http://genopole.toulouse.inra.fr/bioinfo/eugene/EuGeneHom/cgi-bin/EuGeneHom.pl.

Injection by various routes of melanoma antigen-associated macrophages: biodistribution and clinical effects.

Patients' autologous macrophages (AM) were used as antigen-presenting cells (APC) in a vaccination protocol against malignant melanoma. AM were administered by various routes, including intralymphatic, since these cells did not express CCR7, a molecule required for APC migration to lymph nodes. Seven HLA-A2 patients with metastatic melanoma-two classified as M1 and five as M3-were included in the study. AM were produced from leukapheresis-separated mononuclear cells by 7-day culture with granulocyte-macrophage colony-stimulating factor. After separation by elutriation, AM were frozen in aliquots and subsequently thawed at monthly intervals, exposed to MAGE-3(271-279) peptide and injected subcutaneously into lymph nodes or into one peripheral lymph vessel. Intradermal tests were performed before and after treatment to determine peptide reactivity. No acute toxicity was observed following injection. One M1 patient had a 7-mm induration intradermal reaction response and was stabilized for 64 weeks. The M3 patients did not show any immunological or clinical response. In 11 patients, the biodistribution of 111In-labeled AM was investigated. There was no clear evidence that AM injected intradermally or subcutaneously left the site of injection. After injection into a lymph vessel of the foot region, scintigraphs showed five to ten popliteal and inguinocrural lymph nodes. This appeared to be the most efficient way to administer rapidly and safely large amounts of peptide-loaded APC into lymph nodes.

Aceruloplasminemia: new clinical, pathophysiological and therapeutic insights.

Aceruloplasminemia is an autosomal recessive disease of iron overload associated with mutation(s) in the ceruloplasmin gene. We report here a new case of aceruloplasminemia in a woman who is a compound heterozygote for two new mutations. Besides this novel genotypic profile, this observation provides new insights on: (i) iron metabolism with normal erythroid repartition, in the absence of serum non-transferrin-bound iron and with an increase of 59Fe plasma clearance; (ii) hepatic abnormalities associated with the presence of iron-free foci; (iii) the therapeutic management of the disease, chronic subcutaneous infusion of deferrioxamine being remarkably effective at reducing hepatic iron overload.