Construction of 12 EST libraries and characterization of a 12,226 EST dataset for chicory (Cichorium intybus) root, leaves and nodules in the context of carbohydrate metabolism investigation.

BACKGROUND: The industrial chicory, Cichorium intybus, is a member of the Asteraceae family that accumulates fructan of the inulin type in its root. Inulin is a low calories sweetener, a texture agent and a health promoting ingredient due to its prebiotic properties. Average inulin chain length is a critical parameter that is genotype and temperature dependent. In the context of the study of carbohydrate metabolism and to get insight into the transcriptome of chicory root and to visualize temporal changes of gene expression during the growing season, we obtained and characterized 10 cDNA libraries from chicory roots regularly sampled in field during a growing season. A leaf and a nodule libraries were also obtained for comparison. RESULTS: Approximately 1,000 Expressed Sequence Tags (EST) were obtained from each of twelve cDNA libraries resulting in a 12,226 EST dataset. Clustering of these ESTs returned 1,922 contigs and 4,869 singlets for a total of 6,791 putative unigenes. All ESTs were compared to public sequence databases and functionally classified. Data were specifically searched for sequences related to carbohydrate metabolism. Season wide evolution of functional classes was evaluated by comparing libraries at the level of functional categories and unigenes distribution. CONCLUSION: This chicory EST dataset provides a season wide outlook of the genes expressed in the root and to a minor extent in leaves and nodules. The dataset contains more than 200 sequences related to carbohydrate metabolism and 3,500 new ESTs when compared to other recently released chicory EST datasets, probably because of the season wide coverage of the root samples. We believe that these sequences will contribute to accelerate research and breeding of the industrial chicory as well as of closely related species.

Identification of a Nicotiana plumbaginifolia plasma membrane H(+)-ATPase gene expressed in the pollen tube.

In Nicotiana plumbaginifolia, plasma membrane H(+)-ATPases (PMAs) are encoded by a gene family of nine members. Here, we report on the characterization of a new isogene, NpPMA5 (belonging to subfamily IV), and the determination of its expression pattern using the beta-glucuronidase (gusA) reporter gene. pNpPMA5-gusA was expressed in cotyledons, in vascular tissues of the stem (mainly in nodal zones), and in the flower and fruit. In the flower, high expression was found in the pollen tube after in vitro or in vivo germination. Northern blotting analysis confirmed that NpPMA5 was expressed in the pollen tube contrary to NpPMA2 (subfamily I) or NpPMA4 (subfamily II), two genes highly expressed in other tissues. The subcellular localization of PM H(+)-ATPase in the pollen tube was analyzed by immunocytodecoration. As expected, this enzyme was localized to the plasma membrane. However, neither the tip nor the base of the pollen tube was labeled, showing an asymmetrical distribution of this enzyme. This observation supports the hypothesis that the PM H(+)-ATPase is involved in creating the pH gradient that is observed along the pollen tube and is implicated in cell elongation. Compared to other plant PM H(+)-ATPases, the C-terminal region of NpPMA5 is shorter by 26 amino acid residues and is modified in the last 6 residues, due to a sequence rearrangement, which was also found in the orthologous gene of Nicotiana glutinosa, a Nicotiana species distant from N. plumbaginifolia and Petunia hybrida and Lycopersicon esculentum, other Solanacae species. This modification alters part of the PM H(+)-ATPase regulatory domain and raises the question whether this isoform is still regulated.

Complete sequence and comparative genome analysis of the dairy bacterium Streptococcus thermophilus.

The lactic acid bacterium Streptococcus thermophilus is widely used for the manufacture of yogurt and cheese. This dairy species of major economic importance is phylogenetically close to pathogenic streptococci, raising the possibility that it has a potential for virulence. Here we report the genome sequences of two yogurt strains of S. thermophilus. We found a striking level of gene decay (10% pseudogenes) in both microorganisms. Many genes involved in carbon utilization are nonfunctional, in line with the paucity of carbon sources in milk. Notably, most streptococcal virulence-related genes that are not involved in basic cellular processes are either inactivated or absent in the dairy streptococcus. Adaptation to the constant milk environment appears to have resulted in the stabilization of the genome structure. We conclude that S. thermophilus has evolved mainly through loss-of-function events that remarkably mirror the environment of the dairy niche resulting in a severely diminished pathogenic potential.

Identification of regulatory sequence elements within the transcription promoter region of NpABC1, a gene encoding a plant ABC transporter induced by diterpenes.

Expression of NpABC1, a gene encoding a plasma membrane ATP binding cassette (ABC) transporter in Nicotiana plumbaginifolia, is induced by sclareol, an antifungal diterpene produced at the leaf surface, as well as by sclareolide, a close analog. A genomic fragment including the 1282-bp region upstream of the NpABC1 transcription start was fused to the reporter beta-glucuronidase (gus) gene and introduced into N. tabacum BY2 cells for stable transformation. A 25-fold increase in gus expression was observed when cells were treated with sclareolide and some other terpenes. The combined use of 5'-deletion promoter analysis, gel mobility shift assays, DNase I footprinting, and site-directed mutagenesis allowed us to identify three cis-elements (sclareol box 1 (SB1), SB2, and SB3) located, respectively, within nucleotides -827 to -802, -278 to -243, and -216 to -190 upstream of the NpABC1 transcription start. In vivo evaluation of these elements on sclareolide-induced expression showed that mutation of SB1 reduced expression by twofold, while that of SB2 had no effect. On the other hand, SB3 had a marked effect as it completely abolished sclareolide-mediated expression. NpABC1-gus expression was not induced by the stress signals, salicylic acid and ethylene, but was mediated, to some extent, by methyl jasmonate, which is known to promote diterpene synthesis.

Sequence analysis of three Bacillus cereus loci carrying PIcR-regulated genes encoding degradative enzymes and enterotoxin.

PIcR is a pleiotropic regulator of extracellular virulence factors in the opportunistic human pathogen Bacillus cereus and the entomopathogenic Bacillus thuringiensis, and is induced in cells entering stationary phase. Among the genes regulated by PIcR are: pIcA, encoding phosphatidylinositol-specific phospholipase C (PI-PLC); plc, encoding phosphatidylcholine-preferring phospholipase C (PC-PLC); nhe, encoding the non-haemolytic enterotoxin; hbl, encoding haemolytic enterotoxin BL (HBL); and genes specifying a putative S-layer like surface protein and a putative extracellular RNase. By analysing 37.1 kb of DNA sequence surrounding hbl, plcA and plcR, 28 ORFs were predicted. Three novel genes putatively regulated by PlcR and encoding a neutral protease (NprB), a subtilase family serine protease (Sfp) and a putative cell-wall hydrolase (Cwh) were identified. The corresponding sfp and cwh genes were located in the immediate upstream region of plcA and could both be regulated by a putative PlcR-binding site positioned between the inversely transcribed genes. Similarly, nprB was positioned directly upstream and transcribed in the opposite orientation to plcR. Genes surrounding plcA, plcR and hblCDAB that were lacking an upstream PlcR regulatory sequence did not appear to serve functions apparently related to PlcR and did not exhibit a conserved organization in Bacillus subtilis.

A 23911 bp region of the Bacillus subtilis genome comprising genes located upstream and downstream of the lev operon.

Within the framework of the European project to sequence the whole Bacillus subtilis 168 genome, a 23911 bp long chromosomal DNA fragment located around 233 degrees on the B. subtilis genetic map was cloned and sequenced. From the generated sequencing data and the results of the homology search, the primary structure of this region was determined. In addition to the whole lev operon, the region contains putative genes for an amino acid permease, two different alcohol dehydrogenases, a chitosanase, a protein belonging to the LysR family of transcriptional regulators, a protein related to the MerR transcriptional regulator, up to four proteins related to the product of the spoF gene, and genes coding for nine more inferred proteins of unknown function.