Adriana Ravagnani

Spo0A directly controls the switch from acid to solvent production in solvent‐forming clostridia

The spo0A genes of Clostridium beijerinckii NCIMB 8052 and Clostridium cellulolyticum ATCC 35319 were isolated and characterized. The C‐terminal DNA‐binding domains of the predicted products of spo0A from these two organisms, as well as 16 other taxonomically diverse species of Bacillus and Clostridium, show extensive amino acid sequence conservation (56% identity, 65% similarity over 104 residues). A 12‐amino‐acid motif (SRVERAIRHAIE) that forms the putative DNA recognition helix is particularly highly conserved, suggesting a common DNA target. Insertional inactivation of spo0A in C. beijerinckii blocked the formation of solvents (as well as spores and granulose). Sequences resembling Spo0A‐binding motifs (TGNCGAA) are found in the promoter regions of several of the genes whose expression is modulated at the onset of solventogenesis in Clostridium acetobutylicum and C. beijerinckii. These include the upregulated adc gene, encoding acetoacetate decarboxylase (EC 4.1.1.4), and the downregulated ptb gene, encoding phosphotransbutyrylase (EC 2.3.1.c). In vitro gel retardation experiments using C. acetobutylicum adc and C. beijerinckii ptb promoter fragments and recombinant Bacillus subtilis and C. beijerinckii Spo0A suggested that adc and ptb are directly controlled by Spo0A. The binding affinity was reduced when the 0A boxes were destroyed, and enhanced when they were modified to conform precisely to the consensus sequence. In vivo analysis of wild‐type and mutagenized promoters transcriptionally fused to the gusA reporter gene in C. beijerinckii validated this hypothesis. Post‐exponential phase expression from the mutagenized adc promoter was substantially reduced, whereas expression from the mutagenized ptb promoter was not shut down at the end of exponential growth.

Subtle hydrophobic interactions between the seventh residue of the zinc finger loop and the first base of an HGATAR sequence determine promoter-specific recognition by the Aspergillus nidulans GATA factor AreA

A change of a universally conserved leucine to valine in the DNA‐binding domain of the GATA factor AreA results in inability to activate some AreA‐dependent promoters, including that of the uapA gene encoding a specific urate–xanthine permease. Some other AreA‐ dependent promoters become able to function more efficiently than in the wild‐type context. A methionine in the same position results in a less extreme, but opposite effect. Suppressors of the AreA(Val) mutation mapping in the uapA promoter show that the nature of the base in the first position of an HGATAR (where H stands for A, T or C) sequence determines the relative affinity of the promoter for the wild‐type and mutant forms of AreA. In vitro binding studies of wild‐type and mutant AreA proteins are completely consistent with the phenotypes in vivo. Molecular models of the wild‐type and mutant AreA–DNA complexes derived from the atomic coordinates of the GATA‐1–AGATAA complex account both for the phenotypes observed in vivo and the binding differences observed in vitro. Our work extends the consensus of physiologically relevant binding sites from WGATAR to HGATAR, and provides a rationale for the almost universal evolutionary conservation of leucine at the seventh position of the Zn finger of GATA factors. This work shows inter alia that the sequence CGATAGagAGATAA, comprising two almost adjacent AreA‐binding sites, is sufficient to ensure activation of transcription of the uapA gene.

Mutational analysis reveals dispensability of the N-terminal region of the Aspergillus transcription factor mediating nitrogen metabolite repression.

Mutational analysis has enabled identification and localization of an upstream exon of the areA gene of Aspergillus nidulans mediating nitrogen metabolite repression. A mutation in the initiation codon and frameshift mutations, which revert by restoration of the reading frame, established the coding role of the exon and mutations affecting intron splicing in conjunction with DNA sequencing of reverse transcriptase polymerase chain reaction (RT—PCR) products localized the coding region intron. The resulting AREA translation product would have 876 residues. Deletion of the upstream exon such that translation of the remaining areA coding region would yield a protein containing only the 719 C‐terminal residues has only a subtle phenotype, very similar to those resulting from single amino acid replacements in upstream exon‐encoded regions of strong sequence similarity to the Neurospora crassa and Penicillium chrysogenum homologues. A number of areA mRNAs of different sizes are synthesised and appear to be functionally redundant. Synthesis of at least the smallest mRNA(s) is probably subject to autogenous activation. Suppression of frameshift mutations by compensating mutations preventing intron splicing suggests that insertion of a markedly hydrophobic sequence can impair AREA function. Finally, translational initiation for areA can occur within a region of at least 123 nucleotides.

Mutational analysis of the C-terminal region of AREA, the transcription factor mediating nitrogen metabolite repression inAspergillus nidulans

InAspergillus nidulans the positive-acting, wide domain regulatory geneareA mediates nitrogen metabolite repression. Previous analysis demonstrated that the C-terminal 153 residues of theareA product (AREA) are inessential for at least partial expression of most genes subject to regulation byareA. Paradoxically,areAr2, a −1 frameshift replacing the wild-type 122 C-terminal residues with a mutant peptide of 117 amino acids, leads to general loss of function. To determine the basis for theareAr2 mutant phenotype, and as a means of delineating functional domains within the C-terminal region of AREA, we have selected and characterisedareAr2 revertants. Deletion analysis, utilising direct gene replacement, extended this analysis. A mutantareA product truncated immediately after the last residue of the highly conserved GATA (DNA-binding) domain retains partial function. TheareAr2 product retains some function with respect to the expression ofuaZ (encoding urate oxidase) and the mutant allele is partially dominant with respect to nitrate reductase levels. Consistent with theareAr2 product having a debilitating biological activity, we have demonstrated that a polypeptide containing both the wild-type DNA-binding domain and the mutant C-terminus of AREA2 is able to bind DNA in vitro but no longer shows specificity for GATA sequences.

Bioinformatics in the orphan crops

Orphan crops are those which are grown as food, animal feed or other crops of some importance in agriculture, but which have not yet received the investment of research effort or funding required to develop significant public bioinformatics resources. Where an orphan crop is related to a well-characterised model plant species, comparative genomics and bioinformatics can often, though not always, be exploited to assist research and crop improvement. This review addresses some challenges and opportunities presented by bioinformatics in the orphan crops, using three examples: forage grasses from the genera Lolium and Festuca, forage legumes and the second generation energy crop Miscanthus.

Deep Sequencing of Suppression Subtractive Hybridisation Drought and Recovery Libraries of the Non-model Crop Trifolium repens L.

White clover is a short-lived perennial whose persistence is greatly affected by abiotic stresses, particularly drought. The aim of this work was to characterize its molecular response to water deficit and recovery following re-hydration to identify targets for the breeding of tolerant varieties. We created a white clover reference transcriptome of 16,193 contigs by deep sequencing (mean base coverage 387x) four Suppression Subtractive Hybridization (SSH) libraries (a forward and a reverse library for each treatment) constructed from young leaf tissue of white clover at the onset of the response to drought and recovery. Reads from individual libraries were then mapped to the reference transcriptome and processed comparing expression level data. The pipeline generated four robust sets of transcripts induced and repressed in the leaves of plants subjected to water deficit stress (6,937 and 3,142, respectively) and following re-hydration (6,695 and 4,897, respectively). Semi-quantitative polymerase chain reaction was used to verify the expression pattern of 16 genes. The differentially expressed transcripts were functionally annotated and mapped to biological processes and pathways. In agreement with similar studies in other crops, the majority of transcripts up-regulated in response to drought belonged to metabolic processes, such as amino acid, carbohydrate, and lipid metabolism, while transcripts involved in photosynthesis, such as components of the photosystem and the biosynthesis of photosynthetic pigments, were up-regulated during recovery. The data also highlighted the role of raffinose family oligosaccharides (RFOs) and the possible delayed response of the flavonoid pathways in the initial response of white clover to water withdrawal. The work presented in this paper is to our knowledge the first large scale molecular analysis of the white clover response to drought stress and re-hydration. The data generated provide a valuable genomic resource for marker discovery and ultimately for the improvement of white clover.