Mohamed Zouine

The genome sequence of the entomopathogenic bacterium Photorhabdus luminescens.

Photorhabdus luminescens is a symbiont of nematodes and a broad-spectrum insect pathogen. The complete genome sequence of strain TT01 is 5,688,987 base pairs (bp) long and contains 4,839 predicted protein-coding genes. Strikingly, it encodes a large number of adhesins, toxins, hemolysins, proteases and lipases, and contains a wide array of antibiotic synthesizing genes. These proteins are likely to play a role in the elimination of competitors, host colonization, invasion and bioconversion of the insect cadaver, making P. luminescens a promising model for the study of symbiosis and host-pathogen interactions. Comparison with the genomes of related bacteria reveals the acquisition of virulence factors by extensive horizontal transfer and provides clues about the evolution of an insect pathogen. Moreover, newly identified insecticidal proteins may be effective alternatives for the control of insect pests.

Genome sequence of Streptococcus agalactiae, a pathogen causing invasive neonatal disease

Streptococcus agalactiae is a commensal bacterium colonizing the intestinal tract of a significant proportion of the human population. However, it is also a pathogen which is the leading cause of invasive infections in neonates and causes septicaemia, meningitis and pneumonia. We sequenced the genome of the serogroup III strain NEM316, responsible for a fatal case of septicaemia. The genome is 2 211 485 base pairs long and contains 2118 protein coding genes. Fifty‐five per cent of the predicted genes have an ortholog in the Streptococcus pyogenes genome, representing a conserved backbone between these two streptococci. Among the genes in S. agalactiae that lack an ortholog in S. pyogenes , 50% are clustered within 14 islands. These islands contain known and putative virulence genes, mostly encoding surface proteins as well as a number of genes related to mobile elements. Some of these islands could therefore be considered as pathogenicity islands. Compared with other pathogenic streptococci, S. agalactiae shows the unique feature that pathogenicity islands may have an important role in virulence acquisition and in genetic diversity.

Regulatory Features Underlying Pollination-Dependent and -Independent Tomato Fruit Set Revealed by Transcript and Primary Metabolite Profiling

Indole Acetic Acid 9 (IAA9) is a negative auxin response regulator belonging to the Aux/IAA transcription factor gene family whose downregulation triggers fruit set before pollination, thus giving rise to parthenocarpy. In situ hybridization experiments revealed that a tissue-specific gradient of IAA9 expression is established during flower development, the release of which upon pollination triggers the initiation of fruit development. Comparative transcriptome and targeted metabolome analysis uncovered important features of the molecular events underlying pollination-induced and pollination-independent fruit set. Comprehensive transcriptomic profiling identified a high number of genes common to both types of fruit set, among which only a small subset are dependent on IAA9 regulation. The fine-tuning of Aux/IAA and ARF genes and the downregulation of TAG1 and TAGL6 MADS box genes are instrumental in triggering the fruit set program. Auxin and ethylene emerged as the most active signaling hormones involved in the flower-to-fruit transition. However, while these hormones affected only a small number of transcriptional events, dramatic shifts were observed at the metabolic and developmental levels. The activation of photosynthesis and sucrose metabolism-related genes is an integral regulatory component of fruit set process. The combined results allow a far greater comprehension of the regulatory and metabolic events controlling early fruit development both in the presence and absence of pollination/fertilization.

Characteristics of the tomato chromoplast revealed by proteomic analysis

Chromoplasts are non-photosynthetic specialized plastids that are important in ripening tomato fruit (Solanum lycopersicum) since, among other functions, they are the site of accumulation of coloured compounds. Analysis of the proteome of red fruit chromoplasts revealed the presence of 988 proteins corresponding to 802 Arabidopsis unigenes, among which 209 had not been listed so far in plastidial databanks. These data revealed several features of the chromoplast. Proteins of lipid metabolism and trafficking were well represented, including all the proteins of the lipoxygenase pathway required for the synthesis of lipid-derived aroma volatiles. Proteins involved in starch synthesis co-existed with several starch-degrading proteins and starch excess proteins. Chromoplasts lacked proteins of the chlorophyll biosynthesis branch and contained proteins involved in chlorophyll degradation. None of the proteins involved in the thylakoid transport machinery were discovered. Surprisingly, chromoplasts contain the entire set of Calvin cycle proteins including Rubisco, as well as the oxidative pentose phosphate pathway (OxPPP). The present proteomic analysis, combined with available physiological data, provides new insights into the metabolic characteristics of the tomato chromoplast and enriches our knowledge of non-photosynthetic plastids.

Genome sequence of Vibrio splendidus: an abundant planctonic marine species with a large genotypic diversity.

Vibrio splendidus is a dominant Vibrio species in seawater presenting a remarkable genetic diversity; several strains have been linked to invertebrates mortality. We report the complete genome sequence of V. splendidus LGP32, an oyster pathogen, and its comparison with partial genome sequences from related strains. As is typical for the genus, V. splendidus LGP32 contains two chromosomes (3.29 and 1.67 Mb) and most essential cellular processes are encoded by chromosome 1. Comparison with two other V. splendidus partial genome sequences (strains 12B01 and Med222) confirms the previously suggested high genotypic diversity within this species and led to the identification of numerous strain-specific regions that could frequently not be assigned to a specific mechanisms of recombination. Surprisingly, the chromosomal integron, the most variable genetic element in all other Vibrio species analysed to date, is absent from 12B01 and inactivated by a mobile element in Med222, while in LGP32 it only contains a limited number of cassettes. Finally, we found that the LGP32 integron contains a new dfrA cassette, related to those found in resistance integrons of gram-negative clinical isolates. Those results suggest that marine Vibrio can be a source of antibiotic resistance genes.

The SrtA Sortase of Streptococcus agalactiae Is Required for Cell Wall Anchoring of Proteins Containing the LPXTG Motif, for Adhesion to Epithelial Cells, and for Colonization of the Mouse Intestine

ABSTRACT Streptococcus agalactiae (group B streptococcus [GBS]) is the leading cause of neonatal pneumonia, sepsis, and meningitis. An in silico genome analysis indicated that GBS strain NEM316 encodes 35 proteins containing an LPXTG motif which are thought to be covalently linked to the peptidoglycan by an enzyme called sortase. The role of these cell wall-anchored proteins in GBS pathogenesis was evaluated on a global level by inactivating the srtA gene. This gene encodes the major sortase SrtA that anchors most of the LPXTG-containing proteins. We chose the C5a peptidase (ScpB) and Alp2, an abundant immunogenic protein, as prototypical LPXTG-containing proteins. As expected, the SrtA knockout mutant was unable to anchor the C5a peptidase (ScpB) and Alp2 to the cell wall. Complementation with plasmid-borne srtA inserted into the chromosome restored the correct surface localization of both ScpB and Alp2. Interestingly, the SrtA mutant was impaired for binding to the major extracellular matrix components fibronectin and fibrinogen and displayed a significant reduction in adherence to human (A549, HeLa, and Caco-2) and murine (L2) epithelial cells compared to the parental wild-type strain. Surprisingly, the inactivation of srtA had no effect on the virulence of the type III strain of GBS in a neonatal rat model (measured by the 50% lethal dose and lung colonization) but strongly impaired the capacity of the strain to colonize the intestines of gnotobiotic mice in a competition assay. These results demonstrate that LPXTG-containing proteins are involved in cell adhesion and GBS persistence in vivo.

SlARF4, an Auxin Response Factor Involved in the Control of Sugar Metabolism during Tomato Fruit Development

Tomato fruit development is subject to connections between auxin signaling, chloroplastic activity, and sugar metabolism. Successful completion of fruit developmental programs depends on the interplay between multiple phytohormones. However, besides ethylene, the impact of other hormones on fruit quality traits remains elusive. A previous study has shown that down-regulation of SlARF4, a member of the tomato (Solanum lycopersicum) auxin response factor (ARF) gene family, results in a dark-green fruit phenotype with increased chloroplasts (Jones et al., 2002). This study further examines the role of this auxin transcriptional regulator during tomato fruit development at the level of transcripts, enzyme activities, and metabolites. It is noteworthy that the dark-green phenotype of antisense SlARF4-suppressed lines is restricted to fruit, suggesting that SlARF4 controls chlorophyll accumulation specifically in this organ. The SlARF4 underexpressing lines accumulate more starch at early stages of fruit development and display enhanced chlorophyll content and photochemical efficiency, which is consistent with the idea that fruit photosynthetic activity accounts for the elevated starch levels. SlARF4 expression is high in pericarp tissues of immature fruit and then undergoes a dramatic decline at the onset of ripening concomitant with the increase in sugar content. The higher starch content in developing fruits of SlARF4 down-regulated lines correlates with the up-regulation of genes and enzyme activities involved in starch biosynthesis, suggesting their negative regulation by SlARF4. Altogether, the data uncover the involvement of ARFs in the control of sugar content, an essential feature of fruit quality, and provide insight into the link between auxin signaling, chloroplastic activity, and sugar metabolism in developing fruit.

Genome-Wide Identification, Functional Analysis and Expression Profiling of the Aux/IAA Gene Family in Tomato

Auxin is a central hormone that exerts pleiotropic effects on plant growth including the development of roots, shoots, flowers and fruit. The perception and signaling of the plant hormone auxin rely on the cooperative action of several components, among which auxin/indole-3-acetic acid (Aux/IAA) proteins play a pivotal role. In this study, we identified and comprehensively analyzed the entire Aux/IAA gene family in tomato (Solanum lycopersicum), a reference species for Solanaceae plants, and the model plant for fleshy fruit development. Functional characterization using a dedicated single cell system revealed that tomato Aux/IAA proteins function as active repressors of auxin-dependent gene transcription, with, however, different Aux/IAA members displaying varying levels of repression. Phylogenetic analysis indicated that the Aux/IAA gene family is slightly contracted in tomato compared with Arabidopsis, with a lower representation of non-canonical proteins. Sl-IAA genes display distinctive expression pattern in different tomato organs and tissues, and some of them display differential responses to auxin and ethylene, suggesting that Aux/IAAs may play a role in linking both hormone signaling pathways. The data presented here shed more light on Sl-IAA genes and provides new leads towards the elucidation of their function during plant development and in mediating hormone cross-talk.

Functional analysis and binding affinity of tomato ethylene response factors provide insight on the molecular bases of plant differential responses to ethylene.

BackgroundThe phytohormone ethylene is involved in a wide range of developmental processes and in mediating plant responses to biotic and abiotic stresses. Ethylene signalling acts via a linear transduction pathway leading to the activation of Ethylene Response Factor genes (ERF) which represent one of the largest gene families of plant transcription factors. How an apparently simple signalling pathway can account for the complex and widely diverse plant responses to ethylene remains yet an unanswered question. Building on the recent release of the complete tomato genome sequence, the present study aims at gaining better insight on distinctive features among ERF proteins.ResultsA set of 28 cDNA clones encoding ERFs in the tomato (Solanum lycopersicon) were isolated and shown to fall into nine distinct subclasses characterised by specific conserved motifs most of which with unknown function. In addition of being able to regulate the transcriptional activity of GCC-box containing promoters, tomato ERFs are also shown to be active on promoters lacking this canonical ethylene-responsive-element. Moreover, the data reveal that ERF affinity to the GCC-box depends on the nucleotide environment surrounding this cis-acting element. Site-directed mutagenesis revealed that the nature of the flanking nucleotides can either enhance or reduce the binding affinity, thus conferring the binding specificity of various ERFs to target promoters.Based on their expression pattern, ERF genes can be clustered in two main clades given their preferential expression in reproductive or vegetative tissues. The regulation of several tomato ERF genes by both ethylene and auxin, suggests their potential contribution to the convergence mechanism between the signalling pathways of the two hormones.ConclusionsThe data reveal that regions flanking the core GCC-box sequence are part of the discrimination mechanism by which ERFs selectively bind to their target promoters. ERF tissue-specific expression combined to their responsiveness to both ethylene and auxin bring some insight on the complexity and fine regulation mechanisms involving these transcriptional mediators. All together the data support the hypothesis that ERFs are the main component enabling ethylene to regulate a wide range of physiological processes in a highly specific and coordinated manner.

Characterization of the Tomato ARF Gene Family Uncovers a Multi-Levels Post-Transcriptional Regulation Including Alternative Splicing

Background The phytohormone auxin is involved in a wide range of developmental processes and auxin signaling is known to modulate the expression of target genes via two types of transcriptional regulators, namely, Aux/IAA and Auxin Response Factors (ARF). ARFs play a major role in transcriptional activation or repression through direct binding to the promoter of auxin-responsive genes. The present study aims at gaining better insight on distinctive structural and functional features among ARF proteins. Results Building on the most updated tomato (Solanum lycopersicon) reference genome sequence, a comprehensive set of ARF genes was identified, extending the total number of family members to 22. Upon correction of structural annotation inconsistencies, renaming the tomato ARF family members provided a consensus nomenclature for all ARF genes across plant species. In silico search predicted the presence of putative target site for small interfering RNAs within twelve Sl-ARFs while sequence analysis of the 5′-leader sequences revealed the presence of potential small uORF regulatory elements. Functional characterization carried out by transactivation assay partitioned tomato ARFs into repressors and activators of auxin-dependent gene transcription. Expression studies identified tomato ARFs potentially involved in the fruit set process. Genome-wide expression profiling using RNA-seq revealed that at least one third of the gene family members display alternative splicing mode of regulation during the flower to fruit transition. Moreover, the regulation of several tomato ARF genes by both ethylene and auxin, suggests their potential contribution to the convergence mechanism between the signaling pathways of these two hormones. Conclusion All together, the data bring new insight on the complexity of the expression control of Sl-ARF genes at the transcriptional and post-transcriptional levels supporting the hypothesis that these transcriptional mediators might represent one of the main components that enable auxin to regulate a wide range of physiological processes in a highly specific and coordinated manner.