Transcriptional analysis of a Photorhabdus sp. variant reveals transcriptional control of phenotypic variation and multifactorial pathogenicity in insects.

Photorhabdus luminescens lives in a mutualistic association with entomopathogenic nematodes and is pathogenic for insects. Variants of Photorhabdus frequently arise irreversibly and are studied because they have altered phenotypic traits that are potentially important for the host interaction. VAR* is a colonial and phenotypic variant displaying delayed pathogenicity when directly injected into the insect, Spodoptera littoralis. In this study, we evaluated the role of transcriptomic modulation in determining the phenotypic variation and delayed pathogenicity of VAR* with respect to the corresponding wild-type form, TT01α. A P. luminescens microarray identified 148 genes as differentially transcribed between VAR* and TT01α. The net regulator status of VAR* was found to be significantly modified. We also observed in VAR* a decrease in the transcription of genes supporting certain phenotypic traits, such as pigmentation, crystalline inclusion, antibiosis, and protease and lipase activities. Three genes encoding insecticidal toxins (pit and pirB) or putative insecticidal toxins (xnp2) were less transcribed in VAR* than in the TT01α. The overexpression of these genes was not sufficient to restore the virulence of VAR* to the levels of ΤΤ01α, which suggests that the lower virulence of VAR* does not result from impaired toxemia in insects. Three loci involved in oxidative stress responses (sodA, katE, and the hca operon) were found to be downregulated in VAR*. This is consistent with the greater sensitivity of VAR* to H(2)O(2) and may account for the impaired bacteremia in the hemolymph of S. littoralis larvae observed with VAR*. In conclusion, we demonstrate here that some phenotypic traits of VAR* are regulated transcriptionally and highlight the multifactorial nature of pathogenicity in insects.

Early and specific gene expression triggered by rice resistance gene Pi33 in response to infection by ACE1 avirulent blast fungus.

* Our view of genes involved in rice disease resistance is far from complete. Here we used a gene-for-gene relationship corresponding to the interaction between atypical avirulence gene ACE1 from Magnaporthe grisea and rice resistance gene Pi33 to better characterize early rice defence responses induced during such interaction. * Rice genes differentially expressed during early stages of Pi33/ACE1 interaction were identified using DNA chip-based differential hybridization and QRT-PCR survey of the expression of known and putative regulators of disease resistance. * One hundred genes were identified as induced or repressed during rice defence response, 80% of which are novel, including resistance gene analogues. Pi33/ACE1 interaction also triggered the up-regulation of classical PR defence genes and a massive down-regulation of chlorophyll a/b binding genes. Most of these differentially expressed genes were induced or repressed earlier in Pi33/ACE1 interaction than in the gene-for-gene interaction involving Nipponbare resistant cultivar. * Besides demonstrating that an ACE1/Pi33 interaction induced classical and specific expression patterns, this work provides a list of new genes likely to be involved in rice disease resistance.

Whole-genome comparison between Photorhabdus strains to identify genomic regions involved in the specificity of nematode interaction.

The bacterium Photorhabdus establishes a highly specific association with Heterorhabditis, its nematode host. Photorhabdus strains associated with Heterorhabditis bacteriophora or Heterorhabditis megidis were compared using a Photorhabdus DNA microarray. We describe 31 regions belonging to the Photorhabdus flexible gene pool. Distribution analysis of regions among the Photorhabdus genus identified loci possibly involved in nematode specificity.

SUT1 is a putative Zn[II]2Cys6-transcription factor whose upregulation enhances both sterol uptake and synthesis in aerobically growing Saccharomyces cerevisiae cells.

Budding yeast Saccharomyces cerevisiae is a facultative anaerobe whose growth upon oxygen starvation depends on its capacity to import exogenously supplied sterols, whereas the cells are not permeable to these molecules when grown aerobically. Few genes have been identified as being involved in sterol uptake. A higher SUT1 gene dosage leads to a modest, but significant, increase in sterol uptake under aerobic conditions. Based on sequence and physiological data, SUT1 is a hypoxic gene negatively regulated when the cells are grown in the presence of oxygen. We replaced the SUT1 promoter with the constitutive PMA1 gene promoter in order to enhance its transcription. We observed that sterol uptake was then comparable with that obtained with a sterol importing hem1 mutant, although the heme status of the strain was not modified in a process which still occurs when the cells are not growing. Unexpectedly, SUT1 constitutive expression led to a parallel significant increase in endogenous sterol biosynthesis. Moreover, here we present new data showing that the structurally related YPR009 gene (SUT2) is a functional homologue of SUT1, and that both gene products may represent two novel yeast regulatory proteins involved in sterol uptake.

Glycine betaine-assisted protein folding in a lysA mutant of Escherichia coli.

Osmoprotectants exogenously supplied to a hyperosmotic culture medium are efficiently imported and amassed by stressed cells of Escherichia coli. In addition to their evident role in the recovery and maintenance of osmotic balance, these solutes should play an important role on the behavior of cellular macromolecules, for example in the process of protein folding. Using a random chemical mutagenesis approach, a conditional lysine auxotrophic mutant was obtained. The growth of this mutant was restored by addition of either lysine or osmoprotectants including glycine betaine (GB) in the minimal medium. The growth rate increased proportionally with the augmentation of the intracellular GB concentration. The mutation was located in the lysA gene and resulted in the substitution of the Ser at position 384 by Phe of the diaminopimelate decarboxylase (DAPDC), which catalyzes the conversion of meso-diaminopimelate to L-lysine. We purified both the wild type DAPDC and the mutated DAPDC-sf and demonstrated that GB was capable of activating DAPDC-sf in vitro, thus confirming the in vivo results. Most importantly, we showed that the activation was correlated with a conformational change of DAPDC-sf. Taken together, these results show, for the first time, that GB may actively assist in vivo protein folding in a chaperone-like manner.

Isolation and characterization of the Saccharomyces cerevisiae SUT1 gene involved in sterol uptake.

A new gene (SUT1) of Saccharomyces cerevisiae, implicated in sterol uptake, was isolated from a yeast genomic library constructed in a high-copy-number vector by virtue of conferring resistance to fenpropimorph in medium supplemented with ergosterol. The high expression of SUT1 in sterol auxotrophic mutant strains alleviates the requirement for accessory mutations affecting heme biosynthesis and allows sterol uptake in aerobiosis. Measurements of [14C]cholesterol uptake confirmed that SUT1 is involved in sterol absorption. Within the 4.1-kb insert isolated, the functional gene was localised on a 1.7-kb DNA fragment. The nucleotide sequence encodes a predicted protein of 299 amino acids. Northern blot analysis revealed that SUT1 is a new member of the hypoxic gene family. Gene disruption showed that SUT1 is not essential for aerobic or anaerobic yeast growth.