Annick M. Breton

The yeast Rvs161 and Rvs167 proteins are involved in secretory vesicles targeting the plasma membrane and in cell integrity

The Rvs161 and Rvs167 proteins are known to play a role in actin cytokeleton organization and endocytosis. Moreover, Rvs167p functionally interacts with the myosin Myo2p. Therefore, we explored the involvement of the Rvs proteins in vesicle traffic and in cell integrity. The rvs mutants accumulate late secretory vesicles at sites of membrane and cell wall construction. They are synthetic‐lethal with the slt2/mpk1 mutation, which affects the MAP kinase cascade controlled by Pkc1p and is required for cell integrity. The phenotype of the double mutants is close to that described for the pkc1 mutant. Synthetic defects for growth are also observed with mutation in KRE6, a gene coding for a glucan synthase, required for cell wall construction. These data support the idea that the Rvs proteins are involved in the late targeting of vesicles whose cargoes are required for cell wall construction. Copyright

Genetic and functional relationship between Rvsp, myosin and actin in Saccharomyces cerevisiae.

Abstract The rvs mutants display phenotypes close to those described for the actin mutants: disorganization of the actin cytoskeleton, random budding of the diploids, loss of polarity and sensitivity to salt. Mutations in the RVS genes lead to synthetic lethality with a set of mutations in the actin gene, ACT1. This synthetic lethality is allele-specific regarding the act1 mutations, pointing to a region on the actin molecule where contacts with the myosin head have been described. The possible involvement of a myosin in a vital function fulfilled both by the Rvsp proteins and actin is strengthened further by the fact that the double mutants rvs167, myo1 and rvs167, myo2 are lethal and severely affected in growth respectively. These data support the idea that actin, myosin and Rvsp proteins are linked in a common functional pathway in yeast.

Mutants of Myxococcus xanthus impaired in protein secretion: an approach to study of a secretory mechanism

SummaryBacteria able to secrete proteins efficiently into the growth medium occur relatively rarely amongst Gram-negative species. However, the increasing technological interest in protein secretion has focused attention on this process. We have demonstrated that Myxococcus xanthus actively secretes protein. The number of proteins secreted is quite large, but the total amount is strictly regulated and remains constant under conditions that change the specific activities of some of the secreted enzymes. Tn5-insertion mutants were obtained which were impaired in what seems to be the control system for protein secretion. Two of the mutants displayed increased levels of extracellular protein.

Structural instability and stabilization of IncP-1 plasmids integrated into the chromosome of Myxococcus xanthus

Abstract IncP-1 plasmids are self transmissible to Myxococcus xanthus and maintained integrated into the host chromosome where they are liable to structural instability: deletions can span through the integrated plasmid; the frequency of these events depends on the recipient strain and on the localization of the insertion on the chromosome, but not on the structure of the plasmid. It is possible to isolate stabilized insertion, even from one of the most unstable strains, by growing immobilized cells in carrageenan beads in continuous nonselective culture. The remaining resistant cells are stabilized. Both the structural instability and the possible stabilization of the insertion can be useful when IncP-1 plasmids are used as cloning vectors in Myxococcus xanthus .

Production of extracellular native and foreign proteins by immobilized growing cells of Myxococcus xanthus

SummarySeveral strains of the protein-secreting, Gram negative bacterium Myxococcus xanthus were immobilized in carrageenan beads and the production of extracellular proteins was followed.The extracellular proteolytic activity was enhanced and concentrated in the beads. In contrast, the amount of total protein secreted by the cells was not modified by immobilization, but it was also retained and concentrated in the beads, the more, the harder the gel. The amount of slime produced by the cells did not seem to influence protein retention.Foreign proteins expressed from genes cloned in Myxococcus xanthus chromosome can be secreted into the medium by immobilized recombinant strains. A polygalacturonate lyase, expressed from the pelC gene from Erwinia chrysanthemi was only detectable outside of the beads. The pH 2.5 acid phosphatase expressed from the appA gene from Escherichia coli was secreted by immobilized cells at the same rate than did the free cells. It was predominantly found in the medium outside of the beads which represented a first purification and facilitated the continuous production of this protein by immobilized recombinant cells packed in a reactor.

Expression in Myxococcus xanthus of foreign genes coding for secreted pectate lyases of Erwinia chrysanthemi

Structural genes for the five major pectate lyases of Erwinia chrysanthemi, pelA, pelB, pelC, pelD and pelE, have been transferred and expressed in Myxococcus xanthus. These proteins, which are secreted by their original host, are also produced, mainly into the medium, by Myxococcus. These results are discussed in the context of current knowledge about secretion in bacteria.

Mode of insertion of the broad-host-range plasmid RP4 and its derivatives into the chromosome of Myxococcus xanthus.

The mode of insertion of the broad-host-range plasmid RP4 into the chromosome of Myxococcus xanthus strain DZ1 has been analyzed. The plasmid integrated in numerous sites of the chromosome and generated insertional mutations. There is a hot spot of integration located between 31.5 and 34.5 kb clockwise from the EcoRI site of the plasmid. In the absence of this segment the insertion can, however, take place, but much less efficiently. The presence of transposable elements on the plasmid decreases severely the insertion frequency. Once integrated, RP4 could be transferred back to Escherichia coli, either by precise excision or with a segment of the Myxococcus chromosome. The role of site-specific recombination in RP4 integration is discussed.

Overlapping Podospora anserina Transcriptional Responses to Bacterial and Fungal Non Self Indicate a Multilayered Innate Immune Response

Recognition and response to non self is essential to development and survival of all organisms. It can occur between individuals of the same species or between different organisms. Fungi are established models for conspecific non self recognition in the form of vegetative incompatibility (VI), a genetically controlled process initiating a programmed cell death (PCD) leading to the rejection of a fusion cell between genetically different isolates of the same species. In Podospora anserina VI is controlled by members of the hnwd gene family encoding for proteins analogous to NOD Like Receptors (NLR) immune receptors in eukaryotes. It was hypothesized that the hnwd controlled VI reaction was derived from the fungal innate immune response. Here we analyze the P. anserina transcriptional responses to two bacterial species, Serratia fonticola to which P. anserina survives and S. marcescens to which P. anserina succumbs, and compare these to the transcriptional response induced under VI conditions. Transcriptional responses to both bacteria largely overlap, however the number of genes regulated and magnitude of regulation is more important when P. anserina survives. Transcriptional responses to bacteria also overlap with the VI reaction for both up or down regulated gene sets. Genes up regulated tend to be clustered in the genome, and display limited phylogenetic distribution. In all three responses we observed genes related to autophagy to be up-regulated. Autophagy contributes to the fungal survival in all three conditions. Genes encoding for secondary metabolites and histidine kinase signaling are also up regulated in all three conditions. Transcriptional responses also display differences. Genes involved in response to oxidative stress, or encoding small secreted proteins are essentially expressed in response to bacteria, while genes encoding NLR proteins are expressed during VI. Most functions encoded in response to bacteria favor survival of the fungus while most functions up regulated during VI would lead to cell death. These differences are discussed in the frame of a multilayered response to non self in fungi.