Functional study of the Hap4-like genes suggests that the key regulators of carbon metabolism HAP4 and oxidative stress response YAP1 in yeast diverged from a common ancestor.

The transcriptional regulator HAP4, induced by respiratory substrates, is involved in the balance between fermentation and respiration in S. cerevisiae. We identified putative orthologues of the Hap4 protein in all ascomycetes, based only on a conserved sixteen amino acid-long motif. In addition to this motif, some of these proteins contain a DNA-binding motif of the bZIP type, while being nonetheless globally highly divergent. The genome of the yeast Hansenula polymorpha contains two HAP4-like genes encoding the protein HpHap4-A which, like ScHap4, is devoid of a bZIP motif, and HpHap4-B which contains it. This species has been chosen for a detailed examination of their respective properties. Based mostly on global gene expression studies performed in the S. cerevisiae HAP4 disruption mutant (ScΔhap4), we show here that HpHap4-A is functionally equivalent to ScHap4, whereas HpHap4-B is not. Moreover HpHAP4-B is able to complement the H2O2 hypersensitivity of the ScYap1 deletant, YAP1 being, in S. cerevisiae, the main regulator of oxidative stress. Finally, a transcriptomic analysis performed in the ScΔyap1 strain overexpressing HpHAP4-B shows that HpHap4-B acts both on oxidative stress response and carbohydrate metabolism in a manner different from both ScYap1 and ScHap4. Deletion of these two genes in their natural host, H. polymorpha, confirms that HpHAP4-A participates in the control of the fermentation/respiration balance, while HpHAP4-B is involved in oxidative stress since its deletion leads to hypersensitivity to H2O2. These data, placed in an evolutionary context, raise new questions concerning the evolution of the HAP4 transcriptional regulation function and suggest that Yap1 and Hap4 have diverged from a unique regulatory protein in the fungal ancestor.

A general framework for optimization of probes for gene expression microarray and its application to the fungus Podospora anserina.

BACKGROUND: The development of new microarray technologies makes custom long oligonucleotide arrays affordable for many experimental applications, notably gene expression analyses. Reliable results depend on probe design quality and selection. Probe design strategy should cope with the limited accuracy of de novo gene prediction programs, and annotation up-dating. We present a novel in silico procedure which addresses these issues and includes experimental screening, as an empirical approach is the best strategy to identify optimal probes in the in silico outcome. FINDINGS: We used four criteria for in silico probe selection: cross-hybridization, hairpin stability, probe location relative to coding sequence end and intron position. This latter criterion is critical when exon-intron gene structure predictions for intron-rich genes are inaccurate. For each coding sequence (CDS), we selected a sub-set of four probes. These probes were included in a test microarray, which was used to evaluate the hybridization behavior of each probe. The best probe for each CDS was selected according to three experimental criteria: signal-to-noise ratio, signal reproducibility, and representative signal intensities. This procedure was applied for the development of a gene expression Agilent platform for the filamentous fungus Podospora anserina and the selection of a single 60-mer probe for each of the 10,556 P. anserina CDS. CONCLUSIONS: A reliable gene expression microarray version based on the Agilent 44K platform was developed with four spot replicates of each probe to increase statistical significance of analysis.

Global analysis of extracytoplasmic stress signaling in Escherichia coli.

The Bae, Cpx, Psp, Rcs, and sigma(E) pathways constitute the Escherichia coli signaling systems that detect and respond to alterations of the bacterial envelope. Contributions of these systems to stress response have previously been examined individually; however, the possible interconnections between these pathways are unknown. Here we investigate the dynamics between the five stress response pathways by determining the specificities of each system with respect to signal-inducing conditions, and monitoring global transcriptional changes in response to transient overexpression of each of the effectors. Our studies show that different extracytoplasmic stress conditions elicit a combined response of these pathways. Involvement of the five pathways in the various tested stress conditions is explained by our unexpected finding that transcriptional responses induced by the individual systems show little overlap. The extracytoplasmic stress signaling pathways in E. coli thus regulate mainly complementary functions whose discrete contributions are integrated to mount the full adaptive response.

PPIDD: an extraction and visualisation method of biological protein-protein interfaces.

Today, the information for generating reliable protein-protein complex datasets is not directly accessible from PDB structures. Moreover, in X-ray protein structures, different types of contacts can be observed between proteins: contacts in homodimers or inside heterocomplexes considered to be specific, and contacts induced by crystallogenesis processes, considered to be non-specific. However, none of the databases giving access to protein-protein complexes allows the crystallographic interfaces to be distinguished from the biological interfaces. For this reason we developed PPIDD (Protein-Protein Interface Description Database), an innovative tool, which allows the extraction and visualisation of biological protein-protein interfaces from an annotated subset of crystallographic structures of proteins. This tool is focused on the description of protein-protein interfaces corresponding to well-identified classes of protein assemblies. It permits the representation of any of these protein-protein assemblies (duplex) and their interfaces as well as the export of the corresponding molecular structures under a flexible format, which is an extension of the PDBML. Moreover, PPIDD facilitates the construction of subsets of interfaces presenting user-specified common characteristics, to enhance the understanding of the determinants of specific protein-protein interactions.

MAnGO: an interactive R-based tool for two-colour microarray analysis.

UNLABELLED: MAnGO (Microarray Analysis at the Gif/Orsay platform) is an interactive R-based tool for the analysis of two-colour microarray experiments. It is a compilation of various methods, which allows the user (1) to control data quality by detecting biases with a large number of visual representations, (2) to pre-process data (filtering and normalization) and (3) to carry out differential analyses. MAnGO is not only a 'turn-key' tool, oriented towards biologists but also a flexible and adaptable R script oriented towards bioinformaticians. AVAILABILITY: http://bioinfome.cgm.cnrs-gif.fr/.

Different levels of transcriptional regulation due to trophic constraints in the reduced genome of Buchnera aphidicola APS.

Symbiotic associations involving intracellular microorganisms and animals are widespread, especially for species feeding on poor or unbalanced diets. Buchnera aphidicola, the obligate intracellular bacterium associated with most aphid species, provides its hosts with essential amino acids (EAAs), nutrients in short supply in the plant phloem sap. The Buchnera genome has undergone severe reductions during intracellular evolution. Genes for EAA biosynthesis are conserved, but most of the transcriptional regulatory elements are lost. This work addresses two main questions: is transcription in Buchnera (i) regulated and (ii) scaled to aphid EAA demand? Two microarray experiments were designed for profiling the gene expression in Buchnera. The first one was characterized by a specific depletion of tyrosine and phenylalanine in the aphid diet, and the second experiment combined a global diminution of EAAs in the aphid diet with a sucrose concentration increase to manipulate the aphid growth rate. Aphid biological performance and budget analysis (the balance between EAAs provided by the diet and those synthesized by Buchnera) were performed to quantify the nutritional demand from the aphids toward their symbiotic bacteria. Despite the absence of known regulatory elements, a significant transcriptional regulation was observed at different levels of organization in the Buchnera genome: between genes, within putative transcription units, and within specific metabolic pathways. However, unambiguous evidence for transcriptional changes underpinning the scaling of EAA biosynthesis to aphid demand was not obtained. The phenotypic relevance of the transcriptional response from the reduced genome of Buchnera is addressed.

Genome-wide analysis of gene expression in Ralstonia solanacearum reveals that the hrpB gene acts as a regulatory switch controlling multiple virulence pathways.

A 70-mer oligonucleotide-based DNA microarray covering 5,074 of the 5,120 predicted genes from Ralstonia solanacearum has been generated and used to investigate the repertoire of genes that are under the control of the transcription activator HrpB, which governs pathogenicity in this plant pathogenic bacterium. This study identified 143 hrpB up-regulated genes and 50 hrpB down-regulated genes. In addition to extending the repertoire of type III effector proteins with 26 new candidates, this work demonstrates that the hrpB regulon extends beyond type III secretion system-related functions to include a number of genes governing chemotaxy, biosynthesis or catabolism of various low-molecular-weight chemical compounds, and siderophore production and uptake. The presence of several transcripttional regulators and a cluster of genes predicted to encode the synthesis of an acylhomoserine lactone together with the absence of a consensus hrpII box in the promoter of a significant proportion of the hrpB-regulated genes suggest that, for some genes, hrpB regulation might be indirect. Altogether, the data indicate that hrpB acts as a master regulatory gene governing a physiological swing associated with the shift from saprophytic to parasitic life.

Assessment of 35mer amino-modified oligonucleotide based microarray with bacterial samples.

Parallel quantification of a large number of messenger RNA transcripts, using microarray technology, promises to provide unsuspected information about many cellular processes. Although experimental protocols on microarray applications are available, only limited methodological information on glass-slide manufacturing and signal interpretation has been published. The aim of this paper is to provide new insights into the practical aspects of the construction and hybridization of oligonucleotide-based microarrays. The intracellular symbiotic bacterium of aphids, Buchnera aphidicola, is used here as a model organism. The first part of the work is devoted to the optimization of procedures for printing slides, labeling of cDNA targets and hybridization. In the second part, based on a statistical analysis of the results, we discuss the influence of the probe attachment chemistry, of the labeling method, of the oligonucleotide position and of the concentration of a spotted oligonucleotide on signal intensity. The problem of signal specificity is also addressed, based on the calculation of the fluorescent ratio for each probe to its corresponding mismatch control probe. Lastly, the selection of internal spiked RNAs appropriate to our bacterial samples and useful for the data normalization step is presented.

ROSO: optimizing oligonucleotide probes for microarrays.

UNLABELLED: ROSO is software to design optimal oligonucleotide probe sets for microarrays. Selected probes show no significant cross-hybridization, no stable secondary structures and their Tm are chosen to minimize the Tm variability of the probe set. AVAILABILITY: The program is available on the internet. Sources are freely available, for non-profit use, on request to the authors. SUPPLEMENTARY INFORMATION: http://pbil.univ-lyon1.fr/roso