Cell-cell communication in the biocontrol strain Pseudomonas fluorescens CHA0

Summary Pseudomonas fluorescens CHAO is a soil bacterium which was isolated near Morens (Switzerland) and which protects plants from root-pathogenic fungi. This protection is due to extracellular secondary metabolites whose synthesis is regulated by the two-component system GacS/GacA in strain CHAO. Extracellular signals of bacterial origin activate this regulatory system. These signals are different from N-acyl-homoserine lactones, are extracted by dichloromethane and appear to have a low molecular weight. Preliminary evidence was obtained from a small molecule m/z 278 produced by strain CHAO. Similar signals capable of activating GacS/GacA-dependent regulation in strain CHAO were found in a large number of different Gram-negative bacteria. Once activated by signal(s), the sensor GacS is assumed to phosphorylate the response regulator GacA, which positively influences a regulatory cascade, resulting in the synthesis of secondary metabolites. This cascade includes three GacA-controlled small regulatory RNAs and two translational repressor proteins. The regulatory RNAs titrate the repressor proteins; this allows translation of target genes and the synthesis of exoenzymes and secondary metabolites such as antibiotics and hydrogen cyanide. A GFP-based sensor for signal detection was constructed in strain CHAO by fusing the gfp reporter gene to the rsmZ small RNA gene. CHAO mutants defective for signal production were isolated following transposon insertion mutagenesis. In one class of mutants obtained, the gacS gene was inactivated, indicating that GacS/GacA positively controls signal production. In a second class, the thiC gene required for thiamine (vitamin B1) biosynthesis was disrupted. Addition of excess (> 10E-6 M) thiamine to the medium restored signal production. By contrast, when the thiamine concentration was just sufficient to allow normal growth, no production of signal(s) was observed. The mechanism by which thiamine activates signal production remains to be elucidated. Résumé Pseudomonas fluorescens CHAO est une bactérie du sol, isolée près de Morens (Suisse), qui a la capacité de protéger les plantes contre des champignons pathogènes de la racine. Cette protection provient de métabolites secondaires excrétés par la bactérie, dont la synthèse est régulée par le système à deux composants GacS/GacA. Des signaux extracellulaires d'origine bactérienne activent ce système de régulation. Ces signaux, différents des N-acyl¬homosérines lactones, sont extraits par le dichlorométhane et semblent avoir une petite masse moléculaire. Une molécule (masse m/z 278) a été mise en évidence par des expériences préliminaires chez la souche CHAO. Des signaux similaires, capables d'activer la régulation dépendante de GacS/GacA chez la souche CHAO, ont été trouvés chez un grand nombre de bactéries à Gram négative. Une fois activé par le(s) signal(aux), le senseur GacS est supposé phosphoryler le régulateur de réponse GacA, qui influence positivement la cascade de régulation menant à la synthèse des métabolites secondaires. Cette cascade inclut trois petits ARNs régulateurs contrôlés par GacA et deux protéines répresseurs de la traduction. Les ARNs régulateurs titrent les protéines répresseurs, ce qui permet la traduction des gènes cibles et la synthèse d'exoenzymes et de métabolites secondaires tel les antibiotiques et le cyanure d'hydrogène. Un senseur basé sur la GFP pour la détection de signaux a été construit dans la souche CHAO en fusionnant le gène rapporteur gfp au gène de petit ARN rsmZ. Des mutants de CHAO déficients pour la production de signaux ont été isolés au moyen d'une mutagenèse par insertion de transposon. Chez une classe de mutants obtenus, le gène gacS a été inactivé, indiquant que GacS/GacA contrôle positivement la production de signaux. Dans une seconde classe, le gène thiC nécessaire à la biosynthèse de thiamine (vitamine B1) a été interrompu. L'addition en excès (> 10E-6 M) de thiamine au milieu restaure la production de signaux. A l'opposé, quand la concentration de thiamine est juste suffisante pour permettre une croissance normale, aucune production de signaux n'a été observée. Le mécanisme par lequel la thiamine active la production de signaux reste à élucider.

Pyochelin-mediated signalling in Pseudomonas aeruginosa

SUMMARY: Iron is an essential element for nearly all organisms but it is poorly available in most environments and not sufficient to support microbial growth. Bacteria have evolved a range of strategies to acquire this important metal, the most common of these being siderophore-mediated iron uptake. Siderophores are high-affinity iron chelators which are released to the extracellular environment where they complex iron and deliver it to the bacterial cell, via specific uptake systems. The Gram-negative bacterium Pseudomonas aeruginosa produces two siderophores, pyoverdine and pyochelin, which both contribute to the virulence of this opportunistic human pathogen. The genes responsible for pyochelin-mediated iron uptake are grouped in the P. aeruginosa chromosome. The pyochelin biosynthetic genes are organized in two divergent operons, pchDCBA and pchEFGHI, which flank the regulatory gene pchR. The fptA gene, encoding the ferric pyochelin outer membrane receptor, occurs immediately downstream of the pchEFGHI genes. The biosynthesis of the siderophore and its receptor is subjected to dual regulation enabling P. aeruginosa to respond not only to the intracellular iron level but also to the presence of the siderophore in the extracellular environment. Negative regulation is mediated by the widespread Fur protein which employs ferrous iron as a corepressor and binds to a consensus sequence in the promoter region of iron-regulated genes. Positive regulation occurs during iron starvation and requires the AraC-type transcriptional regulator PchR. This regulator, together with pyochelin, induces the expression of pyochelin biosynthesis and uptake genes via a mechanism which was partly unraveled during this thesis. A 32-bp conserved sequence element (PchR-box) was identified in promoter regions of pyochelin-controlled genes. The PchR-box in the pchR-pchDCBA intergenic region was found to be essential for the induction of the pchDCBA operon and for the repression of the divergently transcribed pchR gene. PchR was purified as a fusion with maltose-binding protein (MBP). Mobility shift assays demonstrated specific binding of MBP-PchR to the PchR-box in the presence, but not in the absence of pyochelin. PchR-box mutations which interfered with pyochelin-dependent regulation in vivo, also affected pyochelin-dependent PchR-box recognition in vitro. These results show that pyochelin is the intracellular effector required for PchR-mediated regulation. The fact that extracellular pyochelin triggers this regulation implies that the siderophore can enter the cytoplasm. This conclusion was corroborated by analysing the importance of known and putative pyochelin uptake genes for pyochelin-dependent gene regulation. The pyochelin receptor gene fptA is followed by three genes, fptB, fptC, and fptX, which were shown here to be co-transcribed with fPtA. While fPtX encodes an inner membrane pen-I-lease, the functions of FptB and FptC are currently unknown. FptA and FptX, which are both required for pyochelin-mediated iron uptake, were found to be also needed for pyochelin-dependent gene regulation. FptB and FptC however, were not required and their role, if any, in the uptake of the PchR effector pyochelin remains elusive. RESUME Le fer est un élément essentiel pour la quasi-totalité des organismes, mais dans la plupart des environnements, il est difficilement accessible et insuffisant à la croissance microbienne. Les bactéries ont développé de multiples stratégies pour acquérir ce précieux métal, la plus commune étant l'acquisition au moyen de sidérophores. Les sidérophores sont des petites molécules dotées d'une forte affinité pour le fer qui, une fois relâchées dans l'environnement extracellulaire, vont complexer le fer et le délivrer à la cellule bactérienne par l'intermédiaire de systèmes d'acquisition spécifiques. La bactérie Gram-négative Pseudomonas aeruginosa produit deux sidérophores, la pyoverdine et la pyochéline, qui contribuent également à la virulence de ce pathogène opportuniste. Les gènes impliqués dans l'acquisition du fer à l'aide de la pyochéline sont regroupés sur t. le chromosome de P. aeruginosa. Les gènes de biosynthèse de la pyochéline sont organisés en deux opérons divergents, pchDCBA et pchEFGHI, qui flanquent le gène régulateur pchR. Le gène fptA, codant pour le récepteur de la pyochéline dans la membrane externe, est situé immédiatement en aval des gènes pchEFGHL La biosynthèse du sidérophore et de son récepteur est soumise à une double régulation permettant à P. aeruginosa de réagir non seulement à la quantité de fer intracellulaire, mais également à la présence du sidérophore dans le milieu extracellulaire. La répression se fait par l'intermédiaire de la protéine Fur, qui nécessite le fer ferreux comme co-répresseur et se lie à une séquence consensus dans la région promotrice des gènes régulés par le fer. L'induction se produit lorsque le fer est limitant, et requiert PchR, un régulateur transcriptionnel de la famille AraC. En présence de pyochéline, ce régulateur induit l'expression des gènes de biosynthèse et du récepteur de la pyochéline par l'intermédiaire d'un mécanisme partiellement résolu dans ce travail. Une séquence conservée (PchR-box) a été identifiée dans la région promotrice des gènes régulés par la pyochéline. La PchR-box située dans la région intergénique pchR-pchDCBA s'est révélée être importante pour l'induction de l'opéron pchDCBA et la répression du gène divergent pchR. PchR a été purifiée en tant que protéine de fusion avec une protéine liant le maltose (MBP). Des expériences de gel retard ont démontré la liaison spécifique de la protéine MBP-PchR sur la PchR-box en présence, mais non en absence de pyochéline. Les mutations de la PchR-box qui ont affecté la régulation pyochéline-dépendante in vivo, ont également eu un effet sur la liaison de la protéine in vitro. Ces résultats démontrent que la pyochéline est l'effecteur intracellulaire nécessaire à la régulation par PchR. Le fait que la pyochéline extracellulaire soit capable d'activer cette régulation implique que le sidérophore entre dans le cytoplasme. Cette conclusion a été corroborée par l'évaluation du rôle des gènes connus ou putatifs de l'incorporation du fer via la pyochéline sur la régulation pyochéline-dépendente. Le gène fPtA, codant pour le récepteur de la pyochéline, est suivi de trois gènes, fptB,fptC, et fptX, co-transcrits avec,ffitA. Si sffitX code pour une perméase de la membrane interne, la fonction de FptB et FptC reste obscure. FptA et FptX, nécessaires à l'acquisition du fer par l'intermédiaire de la pyochéline, se sont également révélés être requis pour la régulation pyochéline-dépendante des gènes pchDCBA, pchEFGHI et fptABCX. FptB et FptC n'ont quant à eux vraisemblablement pas de rôle majeur à jouer, si ce n'est aucun, dans l'incorporation de la pyochéline.

Oxidative stress enhances the expression of sulfur assimilation genes: preliminary insights on the Enterococcus faecalis iron-sulfur cluster machinery regulation

The Firmicutes bacteria participate extensively in virulence and pathological processes. Enterococcus faecalis is a commensal microorganism; however, it is also a pathogenic bacterium mainly associated with nosocomial infections in immunocompromised patients. Iron-sulfur [Fe-S] clusters are inorganic prosthetic groups involved in diverse biological processes, whose in vivo formation requires several specific protein machineries. Escherichia coli is one of the most frequently studied microorganisms regarding [Fe-S] cluster biogenesis and encodes the iron-sulfur cluster and sulfur assimilation systems. In Firmicutes species, a unique operon composed of the sufCDSUB genes is responsible for [Fe-S] cluster biogenesis. The aim of this study was to investigate the potential of the E. faecalis sufCDSUB system in the [Fe-S] cluster assembly using oxidative stress and iron depletion as adverse growth conditions. Quantitative real-time polymerase chain reaction demonstrated, for the first time, that Gram-positive bacteria possess an OxyR component responsive to oxidative stress conditions, as fully described for E. coli models. Likewise, strong expression of the sufCDSUB genes was observed in low concentrations of hydrogen peroxide, indicating that the lowest concentration of oxygen free radicals inside cells, known to be highly damaging to [Fe-S] clusters, is sufficient to trigger the transcriptional machinery for prompt replacement of [Fe-S] clusters.

Water-induced modulation of Helicobacter pylori virulence properties

While the influence of water in Helicobacter pylori culturability and membrane integrity has been extensively studied, there are little data concerning the effect of this environment on virulence properties. Therefore, we studied the culturability of water-exposed H. pylori and determined whether there was any relation with the bacterium’s ability to adhere, produce functional components of pathogenicity and induce inflammation and alterations in apoptosis in an experimental model of human gastric epithelial cells. H. pylori partially retained the ability to adhere to epithelial cells even after complete loss of culturability. However, the microorganism is no longer effective in eliciting in vitro host cell inflammation and apoptosis, possibly due to the non-functionality of the cag type IV secretion system. These H. pylori-induced host cell responses, which are lost along with culturability, are known to increase epithelial cell turnover and, consequently, could have a deleterious effect on the initial H. pylori colonisation process. The fact that adhesion is maintained by H. pylori to the detriment of other factors involved in later infection stages appears to point to a modulation of the physiology of the pathogen after water exposure and might provide the microorganism with the necessary means to, at least transiently, colonise the human stomach.

The mazEF toxin-antitoxin system as a novel antibacterial target in Acinetobacter baumannii

Although analysis of toxin-antitoxin (TA) systems can be instructive, to date, there is no information on the prevalence and identity of TA systems based on a large panel of Acinetobacter baumannii clinical isolates. The aim of the current study was to screen for functional TA systems among clinical isolates of A. baumannii and to identify the systems’ locations. For this purpose, we screened 85 A. baumannii isolates collected from different clinical sources for the presence of the mazEF, relBE and higBA TA genes. The results revealed that the genes coding for the mazEF TA system were commonly present in all clinical isolates of A. baumannii. Reverse transcriptase-polymerase chain reaction analysis showed that transcripts were produced in the clinical isolates. Our findings showed that TA genes are prevalent, harboured by chromosomes and transcribed within A. baumannii. Hence, activation of the toxin proteins in the mazEF TA system should be investigated further as an effective antibacterial strategy against this bacterium.

Genome-Wide Analysis of Salicylate and Dibenzofuran Metabolism in Sphingomonas Wittichii RW1.

Sphingomonas wittichii RW1 is a bacterium isolated for its ability to degrade the xenobiotic compounds dibenzodioxin and dibenzofuran (DBF). A number of genes involved in DBF degradation have been previously characterized, such as the dxn cluster, dbfB, and the electron transfer components fdx1, fdx3, and redA2. Here we use a combination of whole genome transcriptome analysis and transposon library screening to characterize RW1 catabolic and other genes implicated in the reaction to or degradation of DBF. To detect differentially expressed genes upon exposure to DBF, we applied three different growth exposure experiments, using either short DBF exposures to actively growing cells or growing them with DBF as sole carbon and energy source. Genome-wide gene expression was examined using a custom-made microarray. In addition, proportional abundance determination of transposon insertions in RW1 libraries grown on salicylate or DBF by ultra-high throughput sequencing was used to infer genes whose interruption caused a fitness loss for growth on DBF. Expression patterns showed that batch and chemostat growth conditions, and short or long exposure of cells to DBF produced very different responses. Numerous other uncharacterized catabolic gene clusters putatively involved in aromatic compound metabolism increased expression in response to DBF. In addition, only very few transposon insertions completely abolished growth on DBF. Some of those (e.g., in dxnA1) were expected, whereas others (in a gene cluster for phenylacetate degradation) were not. Both transcriptomic data and transposon screening suggest operation of multiple redundant and parallel aromatic pathways, depending on DBF exposure. In addition, increased expression of other non-catabolic genes suggests that during initial exposure, S. wittichii RW1 perceives DBF as a stressor, whereas after longer exposure, the compound is recognized as a carbon source and metabolized using several pathways in parallel.

Draft genome sequence of Bacillus thuringiensis 147, a Brazilian strain with high insecticidal activity

Bacillus thuringiensisis a ubiquitous Gram-positive and sporulating bacterium. Its crystals and secreted toxins are useful tools against larvae of diverse insect orders and, as a consequence, an alternative to recalcitrant chemical insecticides. We report here the draft genome sequence ofB. thuringiensis147, a strain isolated from Brazil and with high insecticidal activity. The assembled genome contained 6,167,994 bp and was distributed in seven replicons (a chromosome and 6 plasmids). We identified 12 coding regions, located in two plasmids, which encode insecticidal proteins.

Rickettsia amblyommii infecting Amblyomma sculptum in endemic spotted fever area from southeastern Brazil

The Rickettsia bacteria include the aetiological agents for the human spotted fever (SF) disease. In the present study, a SF groupRickettsia amblyommii related bacterium was detected in a field collected Amblyomma sculptum (Amblyomma cajennense species complex) tick from a Brazilian SF endemic site in southeastern Brazil, in the municipality of Juiz de Fora, state of Minas Gerais. Genetic analysis based on genes ompA,ompB and htrA showed that the detected strain, named R. amblyommii str. JF, is related to the speciesR. amblyommii.

Complete genome of the multidrug-resistant Acinetobacter baumannii strain KBN10P02143 isolated from Korea

Acinetobacter baumannii, a strictly aerobic, non-fermentative, Gram-negative coccobacillary rod-shaped bacterium, is an opportunistic pathogen in humans. We recently isolated a multidrug-resistant A. baumannii strain KBN10P02143 from the pus sample drawn from a surgical patient in South Korea. We report the complete genome of this strain, which consists of 4,139,396 bp (G + C content, 39.08%) with 3,868 protein-coding genes, 73 tRNAs and six rRNA operons. Identification of the genes related to multidrug resistance from this genome and the discovery of a novel conjugative plasmid will increase our understanding of the pathogenicity associated with this species.

Anti-inflammatory properties of secretory IgA on intestinal epithelial cells during infection by Shigella flexneri

The intestinal immune system hasthe complex task to protect the sterilecore of the organism against invasion.Most of invasive enterobacteria targetintestinal epithelial cells (IEC) inducingmajor damages to the mucosa.Shigella flexneri, by invading IECand inducing inflammatory responsesof the colonic mucosa, causes bacillarydysentery, a bloody diarrhea thatis endemic worldwide. The mechanismof entry of this bacterium is stilla matter of debate. Mcells participatingin sampling antigens from the gutlumen through Peyers patches arecommonly considered as the primarysite of entry of the bacteria. Once inthe lamina propria, Shigella can invadeIEC via their basolateral poleand spread from cell-to-cell leading tomassive tissue destruction. More recently,data are accumulating demonstratingthat bacteria can also enter thelamina propria directly via IEC, underscoringIEC as another gate of entry.In addition, the protective role ofsecretory IgA (SIgA) produced byplasmocytes of the lamina propria hasbeen established in shigellosis contextbut few is known about its role inmaintaining IEC monolayer integrity.Here, the impact of the bacterium wasstudied using polarized CaCo 2 cellmonolayer apically infected with avirulent strain of S. flexneri eitheralone or complexed with its cognateanti LPS SIgA. Parameters associatedwith the infection process includingcytokine measurements (IL-8, IL-18)and laser scanning confocal microscopydetection of Zonula Occludens-1, a tight junction (TJ) protein werestudied.We demonstrate that bacteriaare able to infect IEC through theirluminal-like pole as well, inducingthe complete disruption of TJ and thedestruction of the whole reconstitutedCaCo-2 cell monolayer. SIgA uponneutralization of bacteria led to themaintenance of TJ supporting IEC integrity,and the modulation of cytokinereleases. Together with anti-inflammatoryproperties of SIgA, thefact that apical bacteria can damagethe IEC without the intervention ofother cells such as Mcells offers newpossibilities in understanding thepathogenic mechanisms involved inshigellosis.

Development of a multistrain bacterial bioreporter platform for the monitoring of hydrocarbon contaminants in marine environments.

Petroleum hydrocarbons are common contaminants in marine and freshwater aquatic habitats, often occurring as a result of oil spillage. Rapid and reliable on-site tools for measuring the bioavailable hydrocarbon fractions, i.e., those that are most likely to cause toxic effects or are available for biodegradation, would assist in assessing potential ecological damage and following the progress of cleanup operations. Here we examined the suitability of a set of different rapid bioassays (2-3 h) using bacteria expressing the LuxAB luciferase to measure the presence of short-chain linear alkanes, monoaromatic and polyaromatic compounds, biphenyls, and DNA-damaging agents in seawater after a laboratory-scale oil spill. Five independent spills of 20 mL of NSO-1 crude oil with 2 L of seawater (North Sea or Mediterranean Sea) were carried out in 5 L glass flasks for periods of up to 10 days. Bioassays readily detected ephemeral concentrations of short-chain alkanes and BTEX (i.e., benzene, toluene, ethylbenzene, and xylenes) in the seawater within minutes to hours after the spill, increasing to a maximum of up to 80 muM within 6-24 h, after which they decreased to low or undetectable levels. The strong decrease in short-chain alkanes and BTEX may have been due to their volatilization or biodegradation, which was supported by changes in the microbial community composition. Two- and three-ring PAHs appeared in the seawater phase after 24 h with a concentration up to 1 muM naphthalene equivalents and remained above 0.5 muM for the duration of the experiment. DNA-damage-sensitive bioreporters did not produce any signal with the oil-spilled aqueous-phase samples, whereas bioassays for (hydroxy)biphenyls showed occasional responses. Chemical analysis for alkanes and PAHs in contaminated seawater samples supported the bioassay data, but did not show the typical ephemeral peaks observed with the bioassays. We conclude that bacterium-based bioassays can be a suitable alternative for rapid on-site quantitative measurement of hydrocarbons in seawater.

Immunoscintigraphy with antigranulocyte monoclonal antibodies for the diagnosis of septic loosening of hip prostheses.

To determine the value of immunoscintigraphy (IS) with antigranulocyte monoclonal antibodies (Mab) in the diagnosis of subacute or chronic infection of hip prostheses, we prospectively studied 57 patients (23 women and 34 men; age 29-92 years, mean 72.7 years) sent to our institution in the past 6 years for clinical suspicion of septic loosening of a hip prosthesis. Nineteen patients had bilateral prostheses and one of them was studied twice. A total of 78 prostheses were examined. All patients had three-phase bone scans followed by IS with technetium-99m antigranulocyte Mab BW 250/183. Intervals between bone scans and IS varied from 2 days to 4 weeks. Final diagnosis was assessed by culture in 48 cases (articular puncture or intraoperative sampling) and by clinical follow-up of at least 8 months in 30 cases. Twelve prostheses were considered septic and 66 non-septic. The overall sensitivity and specificity were 92% and 64% respectively for bone scans, 67% and 75% for IS and 67% and 84% for both modalities together. In three cases, IS was doubtful and the final clinical diagnosis was negative for infection. False-positive results were observed in the presence of massive loosening of the prosthesis or in association with metaplastic peri-articular bone formation. In three of the four false-negative results, infection was proven only after enrichment of the culture, and the bacterium was Staphylococcus epidermidis. In 12/33 (36%) positive bone scans IS allowed the diagnosis of infection to be excluded. Overall accuracy of both modalities together was 81% and the negative predictive value was 93%, which compares favourably with the results reported for other non-invasive methods.

African tick-bite fever: a new entity in the differential diagnosis of multiple eschars in travelers. Description of five cases imported from South Africa to Switzerland.

African tick-bite fever (ATBF) is a newly described spotted fever rickettsiosis that frequently presents with multiple eschars in travelers returning from sub-Saharan Africa and, to a lesser extent, from the West Indies. It is caused by the bite of an infected Amblyomma tick, whose hunting habits explain the typical presence of multiple inoculation skin lesions and the occurrence of clustered cases. The etiological agent of ATBF is Rickettsia africae, an emerging tick-borne pathogenic bacterium. We describe herein a cluster of five cases of ATBF occurring in Swiss travelers returning from South Africa. The co-incidental infections in these five patients and the presence of multiple inoculation eschars, two features pathognomonic of this rickettsial disease, suggested the diagnosis of ATBF. Indeed, the presence of at least one inoculation eschar is observed in 53-100% of cases and multiple eschars in 21-54%. Two patients presented regional lymphadenitis and one a mild local lymphangitis. Though a cutaneous rash is described in 15-46% of cases, no rash was observed in our series. ATBF was confirmed by serology. Thus, ATBF has recently emerged as one of the most important causes of flu-like illness in travelers returning from Southern Africa. The presence of one or multiple eschars of inoculation is an important clinical clue to the diagnosis. It can be confirmed by serology or by PCR of a biopsy of the eschar. Culture can also be done in reference laboratories. Dermatologists and primary care physicians should know this clinical entity, since an inexpensive and efficient treatment is available.

Antibiotic susceptibility of Estrella lausannensis, a potential emerging pathogen.

Estrella lausannensis is a new Chlamydia-related bacterium, belonging to the Criblamydiaceae family. As suggested by its species name, this bacterium harbors a peculiar star shape. E. lausannensis is able to infect a wide range of amoebal, fish and mammalian cell lines. Moreover, seroprevalence of 2.9% was reported in children and in women with tubal pathology, showing that humans are commonly exposed to this recently discovered strict intracellular bacteria considered as a potential pathogen. Antibiotic susceptibility was determined using two approaches: qPCR and cellular mortality assay. Antibiotics classically used against intracellular bacteria were tested, including β-lactams, fluoroquinolones, cyclines and macrolides. We showed that E. lausannensis is resistant to β-lactams and fluoroquinolones, and sensitive to cyclines. Interestingly, E. lausannensis is slightly resistant to azithromycin with a MIC of 2 μg/ml, which is 10 fold higher compared to Waddlia chondrophila and Parachlamydia acanthamoebae MIC's. A single A2059C mutation in 23S rRNA gene could be responsible for this unexpected resistance.

Sequencing and characterizing the genome of Estrella lausannensis as an undergraduate project: training students and biological insights.

With the widespread availability of high-throughput sequencing technologies, sequencing projects have become pervasive in the molecular life sciences. The huge bulk of data generated daily must be analyzed further by biologists with skills in bioinformatics and by "embedded bioinformaticians," i.e., bioinformaticians integrated in wet lab research groups. Thus, students interested in molecular life sciences must be trained in the main steps of genomics: sequencing, assembly, annotation and analysis. To reach that goal, a practical course has been set up for master students at the University of Lausanne: the "Sequence a genome" class. At the beginning of the academic year, a few bacterial species whose genome is unknown are provided to the students, who sequence and assemble the genome(s) and perform manual annotation. Here, we report the progress of the first class from September 2010 to June 2011 and the results obtained by seven master students who specifically assembled and annotated the genome of Estrella lausannensis, an obligate intracellular bacterium related to Chlamydia. The draft genome of Estrella is composed of 29 scaffolds encompassing 2,819,825 bp that encode for 2233 putative proteins. Estrella also possesses a 9136 bp plasmid that encodes for 14 genes, among which we found an integrase and a toxin/antitoxin module. Like all other members of the Chlamydiales order, Estrella possesses a highly conserved type III secretion system, considered as a key virulence factor. The annotation of the Estrella genome also allowed the characterization of the metabolic abilities of this strictly intracellular bacterium. Altogether, the students provided the scientific community with the Estrella genome sequence and a preliminary understanding of the biology of this recently-discovered bacterial genus, while learning to use cutting-edge technologies for sequencing and to perform bioinformatics analyses.