Evidence for the involvement of the anthranilate degradation pathway in Pseudomonas aeruginosa biofilm formation.

Bacterial biofilms are complex cell communities found attached to surfaces and surrounded by an extracellular matrix composed of exopolysaccharides, DNA, and proteins. We investigated the whole-genome expression profile of Pseudomonas aeruginosa sessile cells (SCs) present in biofilms developed on a glass wool substratum. The transcriptome and proteome of SCs were compared with those of planktonic cell cultures. Principal component analysis revealed a biofilm-specific gene expression profile. Our study highlighted the overexpression of genes controlling the anthranilate degradation pathway in the SCs grown on glass wool for 24 h. In this condition, the metabolic pathway that uses anthranilate for Pseudomonas quinolone signal production was not activated, which suggested that anthranilate was primarily being consumed for energy metabolism. Transposon mutants defective for anthranilate degradation were analyzed in a simple assay of biofilm formation. The phenotypic analyses confirmed that P. aeruginosa biofilm formation partially depended on the activity of the anthranilate degradation pathway. This work points to a new feature concerning anthranilate metabolism in P. aeruginosa SCs.

Proteome analysis of a CTR9 deficient yeast strain suggests that Ctr9 has function(s) independent of the Paf1 complex.

The Ctr9 protein is a member of the Paf1 complex implicated in multiple functions: transcription initiation and elongation by RNA pol II, RNA processing and histone modifications. It has also been described as a triple-helical DNA binding protein. Loss of Ctr9 results in severe phenotypes similar to the loss of Paf1p, a Paf1 complex subunit. However, the exact role of Ctr9 is not entirely established. To study the biological role of the protein Ctr9 in yeast, we used 2-D gel electrophoresis and characterized proteome alterations in a ctr9Δ mutant strain. Here we present results suggesting that Ctr9 has function distinct from its established role in the Paf1 complex. This role could be linked to its ability to bind to DNA complex structures as triplexes that may have function in regulation of gene expression.

Exploring the dynamics of the yeast proteome by means of 2-DE.

In this study we combined pulse chase experiments and 2-DE in order to investigate how newly synthesized proteins are processed or modified to yield a functional yeast proteome. This approach allowed us to follow the fate of 560 native yeast proteins from the time they were synthesized up to several hours later. Among these, 81 were observed to vary during the chase, either increasing or decreasing. In addition, 60 were found to be modified immediately after their synthesis. Taking advantage of protein identifications, we characterized a wide variety of post-translational events responsible for these changes, such as protein turnover, protein maturation and different types of PTMs. These events operate over very different time scales, ranging from the brief period required for co-translational modifications to one generation time or more. In light of these results, the functional proteome of exponentially growing cells appears to be the product of a permanent remodelling process that modifies native proteins far beyond the time they have been synthesized. This study also allowed us to obtain information on the half-lives of 260 abundant yeast proteins.

Yeast proteome map (last update).

The identification of proteins separated on 2-D gels is essential to exploit the full potential of 2-D gel electrophoresis for proteomic investigations. For this purpose we have undertaken the systematic identification of Saccharomyces cerevisiae proteins separated on 2-D gels. We report here the identification by mass spectrometry of 100 novel yeast protein spots that have so far not been tackled due to their scarcity on our standard 2-D gels. These identifications extend the number of protein spots identified on our yeast 2-D proteome map to 716. They correspond to 485 unique proteins. Among these, 154 were resolved into several isoforms. The present data set can now be expanded to report for the first time a map of 363 protein isoforms that significantly deepens our knowledge of the yeast proteome. The reference map and a list of all identified proteins can be accessed on the Yeast Protein Map server (www.ibgc.u-bordeaux2.fr/YPM).

The yeast PNC1 longevity gene is up-regulated by mRNA mistranslation.

Translation fidelity is critical for protein synthesis and to ensure correct cell functioning. Mutations in the protein synthesis machinery or environmental factors that increase synthesis of mistranslated proteins result in cell death and degeneration and are associated with neurodegenerative diseases, cancer and with an increasing number of mitochondrial disorders. Remarkably, mRNA mistranslation plays critical roles in the evolution of the genetic code, can be beneficial under stress conditions in yeast and in Escherichia coli and is an important source of peptides for MHC class I complex in dendritic cells. Despite this, its biology has been overlooked over the years due to technical difficulties in its detection and quantification. In order to shed new light on the biological relevance of mistranslation we have generated codon misreading in Saccharomyces cerevisiae using drugs and tRNA engineering methodologies. Surprisingly, such mistranslation up-regulated the longevity gene PNC1. Similar results were also obtained in cells grown in the presence of amino acid analogues that promote protein misfolding. The overall data showed that PNC1 is a biomarker of mRNA mistranslation and protein misfolding and that PNC1-GFP fusions can be used to monitor these two important biological phenomena in vivo in an easy manner, thus opening new avenues to understand their biological relevance.

Sequence requirements for Nalpha-terminal acetylation of yeast proteins by NatA.

NatA is the major N-terminal acetyltransferase of the yeast Saccharomyces cerevisiae. In this study, we took advantage of our recent data on N-terminal acetylation of proteins of the yeast protein map to update the list of proteins with known NatA-dependent acetylation status. Furthermore, using the information available on the acetylation status of 100 novel proteins, we re-examined the rules for acetylation by NatA. The results refine our previous knowledge on NatA substrate specificity depending on the N-terminal and penultimate residues. In particular, we found that the acetylation frequencies of Ser-, Thr- and Ala-, the three residues most often acetylated by NatA, are higher than previously reported. In addition, comparison of the N-terminal region of acetylated and non-acetylated proteins revealed differences in amino acid composition that extend over the 25 first amino acid residues: acetylated proteins are characterized by a higher frequency of glutamate and glutamine and a lower frequency of lysine, arginine and histidine. We suggest that the particularities in amino acid composition of the N-terminal region of acetylated proteins facilitate its interaction with the Nat1p subunit of NatA and its guidance to the catalytic subunit Ard1p.

Critical roles for a genetic code alteration in the evolution of the genus Candida.

During the last 30 years, several alterations to the standard genetic code have been discovered in various bacterial and eukaryotic species. Sense and nonsense codons have been reassigned or reprogrammed to expand the genetic code to selenocysteine and pyrrolysine. These discoveries highlight unexpected flexibility in the genetic code, but do not elucidate how the organisms survived the proteome chaos generated by codon identity redefinition. In order to shed new light on this question, we have reconstructed a Candida genetic code alteration in Saccharomyces cerevisiae and used a combination of DNA microarrays, proteomics and genetics approaches to evaluate its impact on gene expression, adaptation and sexual reproduction. This genetic manipulation blocked mating, locked yeast in a diploid state, remodelled gene expression and created stress cross-protection that generated adaptive advantages under environmental challenging conditions. This study highlights unanticipated roles for codon identity redefinition during the evolution of the genus Candida, and strongly suggests that genetic code alterations create genetic barriers that speed up speciation.

Yeast proteome map (update 2006).

To improve the potential of two-dimensional gel electrophoresis for proteomic investigations in yeast we have undertaken the systematic identification of Saccharomyces cerevisiae proteins separated on 2-D gels. We report here the identification of 187 novel protein spots. They were identified by two methods, mass spectrometry and gene inactivation. These identifications extend the number of protein spots identified on our yeast 2-D proteome map to 602, i.e. nearly half the detectable spots of the proteome map. These spots correspond to 417 different proteins. The reference map and the list of identified proteins can be accessed on the Yeast Protein Map server (www.ibgc.u-bordeaux2.fr/YPM).

High cAMP levels antagonize the reprogramming of gene expression that occurs at the diauxic shift in Saccharomyces cerevisiae.

In order to analyse the involvement of the cAMP pathway in the regulation of gene expression in Saccharomyces cerevisiae, we have examined the effect of cAMP on protein synthesis by using two-dimensional gel electrophoresis. cAMP had only a minor effect on the protein pattern of cells growing exponentially on glucose. However, it interfered with the changes in gene expression normally occurring upon glucose exhaustion in yeast cultures, maintaining a protein pattern typical of cells growing on glucose. This effect was accompanied by a delay before growth recovery on ethanol. We propose a model in which the cAMP-signalling pathway has a role in the maintenance of gene expression, rather than in the determination of a specific programme. A decrease of cAMP would then be required for metabolic transitions such as the diauxic phase.