Thomas Junier

Phylogenetic and functional marker genes to study ammonia-oxidizing microorganisms (AOM) in the environment

The oxidation of ammonia plays a significant role in the transformation of fixed nitrogen in the global nitrogen cycle. Autotrophic ammonia oxidation is known in three groups of microorganisms. Aerobic ammonia-oxidizing bacteria and archaea convert ammonia into nitrite during nitrification. Anaerobic ammonia-oxidizing bacteria (anammox) oxidize ammonia using nitrite as electron acceptor and producing atmospheric dinitrogen. The isolation and cultivation of all three groups in the laboratory are quite problematic due to their slow growth rates, poor growth yields, unpredictable lag phases, and sensitivity to certain organic compounds. Culture-independent approaches have contributed importantly to our understanding of the diversity and distribution of these microorganisms in the environment. In this review, we present an overview of approaches that have been used for the molecular study of ammonia oxidizers and discuss their application in different environments.

Genome-wide search reveals a novel GacA-regulated small RNA in Pseudomonas species

BackgroundSmall RNAs (sRNAs) are widespread among bacteria and have diverse regulatory roles. Most of these sRNAs have been discovered by a combination of computational and experimental methods. In Pseudomonas aeruginosa, a ubiquitous Gram-negative bacterium and opportunistic human pathogen, the GacS/GacA two-component system positively controls the transcription of two sRNAs (RsmY, RsmZ), which are crucial for the expression of genes involved in virulence. In the biocontrol bacterium Pseudomonas fluorescens CHA0, three GacA-controlled sRNAs (RsmX, RsmY, RsmZ) regulate the response to oxidative stress and the expression of extracellular products including biocontrol factors. RsmX, RsmY and RsmZ contain multiple unpaired GGA motifs and control the expression of target mRNAs at the translational level, by sequestration of translational repressor proteins of the RsmA family.ResultsA combined computational and experimental approach enabled us to identify 14 intergenic regions encoding sRNAs in P. aeruginosa. Eight of these regions encode newly identified sRNAs. The intergenic region 1698 was found to specify a novel GacA-controlled sRNA termed RgsA. GacA regulation appeared to be indirect. In P. fluorescens CHA0, an RgsA homolog was also expressed under positive GacA control. This 120-nt sRNA contained a single GGA motif and, unlike RsmX, RsmY and RsmZ, was unable to derepress translation of the hcnA gene (involved in the biosynthesis of the biocontrol factor hydrogen cyanide), but contributed to the bacteriums resistance to hydrogen peroxide. In both P. aeruginosa and P. fluorescens the stress sigma factor RpoS was essential for RgsA expression.ConclusionThe discovery of an additional sRNA expressed under GacA control in two Pseudomonas species highlights the complexity of this global regulatory system and suggests that the mode of action of GacA control may be more elaborate than previously suspected. Our results also confirm that several GGA motifs are required in an sRNA for sequestration of the RsmA protein.

TRiFLe, a program for in silico terminal restriction fragment length polymorphism analysis with user-defined sequence sets

ABSTRACT We describe TRiFLe, a freely accessible computer program that generates theoretical terminal restriction fragments (T-RFs) from any user-supplied sequence set tailored to a particular group of organisms, sequences from clone libraries, or sequences from specific genes. The program allows a rapid identification of the most polymorphic enzymes, creates a collection of T-RFs for the data set, and can potentially identify specific T-RFs in T-RF length polymorphism (T-RFLP) patterns by comparing theoretical and experimental results. TRiFLE was used for analyzing T-RFLP data generated for the amoA and pmoA genes. The peaks identified in the T-RFLP patterns show an overlap of ammonia- and methane-oxidizing bacteria in the metalimnion of a subtropical lake.

Dotlet: diagonal plots in a Web browser

UNLABELLEDnDotlet is a program for comparing sequences by the diagonal plot method. It is designed to be platform-independent and to run in a Web browser, thus enabling the majority of researchers to use it.nnnAVAILABILITYnThe applet can be tested at http://www.isrec.isb-sib.ch/java/dotlet/ Dotlet.html, and the source code is available upon request.nnnCONTACTnThomas. Junier Marco.Pagni @isrec.unil.ch SUPPLEMENTARY: The full documentation about d o t l e t is available from the above URL.

New complete genome sequences of human rhinoviruses shed light on their phylogeny and genomic features

BackgroundHuman rhinoviruses (HRV), the most frequent cause of respiratory infections, include 99 different serotypes segregating into two species, A and B. Rhinoviruses share extensive genomic sequence similarity with enteroviruses and both are part of the picornavirus family. Nevertheless they differ significantly at the phenotypic level. The lack of HRV full-length genome sequences and the absence of analysis comparing picornaviruses at the whole genome level limit our knowledge of the genomic features supporting these differences.ResultsHere we report complete genome sequences of 12 HRV-A and HRV-B serotypes, more than doubling the current number of available HRV sequences. The whole-genome maximum-likelihood phylogenetic analysis suggests that HRV-B and human enteroviruses (HEV) diverged from the last common ancestor after their separation from HRV-A. On the other hand, compared to HEV, HRV-B are more related to HRV-A in the capsid and 3B-C regions. We also identified the presence of a 2C cis-acting replication element (cre) in HRV-B that is not present in HRV-A, and that had been previously characterized only in HEV. In contrast to HEV viruses, HRV-A and HRV-B share also markedly lower GC content along the whole genome length.ConclusionOur findings provide basis to speculate about both the biological similarities and the differences (e.g. tissue tropism, temperature adaptation or acid lability) of these three groups of viruses.

Rhinovirus genome evolution during experimental human infection.

Human rhinoviruses (HRVs) evolve rapidly due in part to their error-prone RNA polymerase. Knowledge of the diversity of HRV populations emerging during the course of a natural infection is essential and represents a basis for the design of future potential vaccines and antiviral drugs. To evaluate HRV evolution in humans, nasal wash samples were collected daily for five days from 15 immunocompetent volunteers experimentally infected with a reference stock of HRV-39. In parallel, HeLa-OH cells were inoculated to compare HRV evolution in vitro. Nasal wash in vivo assessed by real-time PCR showed a viral load that peaked at 48–72 h. Ultra-deep sequencing was used to compare the low-frequency mutation populations present in the HRV-39 inoculum in two human subjects and one HeLa-OH supernatant collected 5 days post-infection. The analysis revealed hypervariable mutation locations in VP2, VP3, VP1, 2C and 3C genes and conserved regions in VP4, 2A, 2B, 3A, 3B and 3D genes. These results were confirmed by classical sequencing of additional samples, both from inoculated volunteers and independent cell infections, and suggest that HRV inter-host transmission is not associated with a strong bottleneck effect. A specific analysis of the VP1 capsid gene of 15 human cases confirmed the high mutation incidence in this capsid region, but not in the antiviral drug-binding pocket. We could also estimate a mutation frequency in vivo of 3.4×10−4 mutations/nucleotides and 3.1×10−4 over the entire ORF and VP1 gene, respectively. In vivo, HRV generate new variants rapidly during the course of an acute infection due to mutations that accumulate in hot spot regions located at the capsid level, as well as in 2C and 3C genes.

The genome of the Gram-positive metal- and sulfate-reducing bacterium Desulfotomaculum reducens strain MI-1

Spore-forming, Gram-positive sulfate-reducing bacteria (SRB) represent a group of SRB that dominates the deep subsurface as well as niches in which resistance to oxygen and dessication is an advantage. Desulfotomaculum reducens strain MI-1 is one of the few cultured representatives of that group with a complete genome sequence available. The metabolic versatility of this organism is reflected in the presence of genes encoding for the oxidation of various electron donors, including three- and four-carbon fatty acids and alcohols. Synteny in genes involved in sulfate reduction across all four sequenced Gram-positive SRB suggests a distinct sulfate-reduction mechanism for this group of bacteria. Based on the genomic information obtained for sulfate reduction in D. reducens, the transfer of electrons to the sulfite and APS reductases is proposed to take place via the quinone pool and heterodisulfide reductases respectively. In addition, both H(2) -evolving and H(2) -consuming cytoplasmic hydrogenases were identified in the genome, pointing to potential cytoplasmic H(2) cycling in the bacterium. The mechanism of metal reduction remains unknown.

Rhinovirus Genome Variation during Chronic Upper and Lower Respiratory Tract Infections

Routine screening of lung transplant recipients and hospital patients for respiratory virus infections allowed to identify human rhinovirus (HRV) in the upper and lower respiratory tracts, including immunocompromised hosts chronically infected with the same strain over weeks or months. Phylogenetic analysis of 144 HRV-positive samples showed no apparent correlation between a given viral genotype or species and their ability to invade the lower respiratory tract or lead to protracted infection. By contrast, protracted infections were found almost exclusively in immunocompromised patients, thus suggesting that host factors rather than the virus genotype modulate disease outcome, in particular the immune response. Complete genome sequencing of five chronic cases to study rhinovirus genome adaptation showed that the calculated mutation frequency was in the range observed during acute human infections. Analysis of mutation hot spot regions between specimens collected at different times or in different body sites revealed that non-synonymous changes were mostly concentrated in the viral capsid genes VP1, VP2 and VP3, independent of the HRV type. In an immunosuppressed lung transplant recipient infected with the same HRV strain for more than two years, both classical and ultra-deep sequencing of samples collected at different time points in the upper and lower respiratory tracts showed that these virus populations were phylogenetically indistinguishable over the course of infection, except for the last month. Specific signatures were found in the last two lower respiratory tract populations, including changes in the 5′UTR polypyrimidine tract and the VP2 immunogenic site 2. These results highlight for the first time the ability of a given rhinovirus to evolve in the course of a natural infection in immunocompromised patients and complement data obtained from previous experimental inoculation studies in immunocompetent volunteers.

Contribution of Genetic Background, Traditional Risk Factors, and HIV-Related Factors to Coronary Artery Disease Events in HIV-Positive Persons

BACKGROUNDnPersons infected with human immunodeficiency virus (HIV) have increased rates of coronary artery disease (CAD). The relative contribution of genetic background, HIV-related factors, antiretroviral medications, and traditional risk factors to CAD has not been fully evaluated in the setting of HIV infection.nnnMETHODSnIn the general population, 23 common single-nucleotide polymorphisms (SNPs) were shown to be associated with CAD through genome-wide association analysis. Using the Metabochip, we genotyped 1875 HIV-positive, white individuals enrolled in 24 HIV observational studies, including 571 participants with a first CAD event during the 9-year study period and 1304 controls matched on sex and cohort.nnnRESULTSnA genetic risk score built from 23 CAD-associated SNPs contributed significantly to CAD (P = 2.9 × 10(-4)). In the final multivariable model, participants with an unfavorable genetic background (top genetic score quartile) had a CAD odds ratio (OR) of 1.47 (95% confidence interval [CI], 1.05-2.04). This effect was similar to hypertension (OR = 1.36; 95% CI, 1.06-1.73), hypercholesterolemia (OR = 1.51; 95% CI, 1.16-1.96), diabetes (OR = 1.66; 95% CI, 1.10-2.49), ≥ 1 year lopinavir exposure (OR = 1.36; 95% CI, 1.06-1.73), and current abacavir treatment (OR = 1.56; 95% CI, 1.17-2.07). The effect of the genetic risk score was additive to the effect of nongenetic CAD risk factors, and did not change after adjustment for family history of CAD.nnnCONCLUSIONSnIn the setting of HIV infection, the effect of an unfavorable genetic background was similar to traditional CAD risk factors and certain adverse antiretroviral exposures. Genetic testing may provide prognostic information complementary to family history of CAD.

trEST, trGEN and Hits: access to databases of predicted protein sequences

High throughput genome (HTG) and expressed sequence tag (EST) sequences are currently the most abundant nucleotide sequence classes in the public database. The large volume, high degree of fragmentation and lack of gene structure annotations prevent efficient and effective searches of HTG and EST data for protein sequence homologies by standard search methods. Here, we briefly describe three newly developed resources that should make discovery of interesting genes in these sequence classes easier in the future, especially to biologists not having access to a powerful local bioinformatics environment. trEST and trGEN are regularly regenerated databases of hypothetical protein sequences predicted from EST and HTG sequences, respectively. Hits is a web-based data retrieval and analysis system providing access to precomputed matches between protein sequences (including sequences from trEST and trGEN) and patterns and profiles from Prosite and Pfam. The three resources can be accessed via the Hits home page (http://hits. isb-sib.ch).