Small RNA Deep-Sequencing Analyses Reveal a New Regulator of Virulence in Agrobacterium fabrum C58.

Novel ways of regulating Ti plasmid functions were investigated by studying small RNAs (sRNAs) that are known to act as posttranscriptional regulators in plant pathogenic bacteria. sRNA-seq analyses of Agrobacterium fabrum C58 allowed us to identify 1,108 small transcripts expressed in several growth conditions that could be sRNAs. A quarter of them were confirmed by bioinformatics or by biological experiments. Antisense RNAs represent 24% of the candidates and they are over-represented on the pTi (with 62% of pTi sRNAs), suggesting differences in the regulatory mechanisms between the essential and accessory replicons. Moreover, a large number of these pTi antisense RNAs are transcribed opposite to those genes involved in virulence. Others are 5'- and 3'-untranslated region RNAs and trans-encoded RNAs. We have validated, by rapid amplification of cDNA ends polymerase chain reaction, the transcription of 14 trans-encoded RNAs, among which RNA1111 is expressed from the pTiC58. Its deletion decreased the aggressiveness of A. fabrum C58 on tomatoes, tobaccos, and kalanchoe, suggesting that this sRNA activates virulence. The identification of its putative target mRNAs (6b gene, virC2, virD3, and traA) suggests that this sRNA may coordinate two of the major pTi functions, the infection of plants and its dissemination among bacteria.

Dicer-2- and Piwi-mediated RNA interference in Rift Valley fever virus-infected mosquito cells.

Rift Valley fever virus (RVFV) is a Phlebovirus (Bunyaviridae family) transmitted by mosquitoes. It infects humans and ruminants, causing dramatic epidemics and epizootics in Africa, Yemen, and Saudi Arabia. While recent studies demonstrated the importance of the nonstructural protein NSs as a major component of virulence in vertebrates, little is known about infection of mosquito vectors. Here we studied RVFV infection in three different mosquito cell lines, Aag2 cells from Aedes aegypti and U4.4 and C6/36 cells from Aedes albopictus. In contrast with mammalian cells, where NSs forms nuclear filaments, U4.4 and Aag2 cells downregulated NSs expression such that NSs filaments were never formed in nuclei of U4.4 cells and disappeared at an early time postinfection in the case of Aag2 cells. On the contrary, in C6/36 cells, NSs nuclear filaments were visible during the entire time course of infection. Analysis of virus-derived small interfering RNAs (viRNAs) by deep sequencing indicated that production of viRNAs was very low in C6/36 cells, which are known to be Dicer-2 deficient but expressed some viRNAs presenting a Piwi signature. In contrast, Aag2 and U4.4 cells produced large amounts of viRNAs predominantly matching the S segment and displaying Dicer-2 and Piwi signatures. Whereas 21-nucleotide (nt) Dicer-2 viRNAs were prominent during early infection, the population of 24- to 27-nt Piwi RNAs (piRNAs) increased progressively and became predominant later during the acute infection and during persistence. In Aag2 and U4.4 cells, the combined actions of the Dicer-2 and Piwi pathways triggered an efficient antiviral response permitting, among other actions, suppression of NSs filament formation and allowing establishment of persistence. In C6/36 cells, Piwi-mediated RNA interference (RNAi) appeared to be sufficient to mount an antiviral response against a secondary infection with a superinfecting virus. This study provides new insights into the role of Dicer and Piwi in mosquito antiviral defense and the development of the antiviral response in mosquitoes.

S-adenosylmethionine decarboxylase of Bacillus subtilis is closely related to archaebacterial counterparts.

Bacillus subtilis synthesizes polyamines by decarboxylating arginine to agmatine, which is subsequently hydrolysed to putrescine. Spermidine is synthesized from putrescine and decarboxylated S-adenosylmethionine (dAdoMet). In Gram-negative bacteria and in eukaryotes, AdoMet is decarboxylated by an unusual 'pyruvoyl' AdoMet decarboxylase (SpeD), the catalytic pyruvoyl moiety of which is generated by serinolysis of an internal serine with self-cleavage of the protein at the upstream peptide bond. Neither the Gram-positive bacterial nor the archaeal counterpart of the Escherichia coli SpeD enzyme were known. We have identified the corresponding B. subtilis speD gene (formely ytcF). Heterologous expression of the cognate Methanococcus jannaschii protein, MJ0315, demonstrated that it displays the same activity as B. subtilis SpeD, indicating that spermidine biosynthesis in Gram-positive bacteria and in archaea follows a pathway very similar to that of Gram-negatives and eukarya. In B. subtilis, transcription of speD is modulated by spermidine and methionine. Its expression is high under usual growth conditions. In contrast, the SpeD protein self-cleaves slowly in vitro, a noticeable difference with its archaeal counterpart. Under certain growth conditions (minimal medium containing succinate and glutamate as a carbon source), speD is co-transcribed with gapB, the gene encoding glyceraldehyde-3-phosphate dehydrogenase, an enzyme required for gluconeogenesis. This observation may couple polyamine metabolism to sulphur and carbon metabolism by a so far unknown mechanism.

Large-scale phenotypic analysis--the pilot project on yeast chromosome III.

In 1993, a pilot project for the functional analysis of newly discovered open reading frames, presumably coding for proteins, from yeast chromosome III was launched by the European Community. In the frame of this programme, we have developed a large-scale screening for the identification of gene/protein functions via systematic phenotypic analysis. To this end, some 80 haploid mutant yeast strains were constructed, each carrying a targeted deletion of a single gene obtained by HIS3 or TRP1 transplacement in the W303 background and a panel of some 100 growth conditions was established, ranging from growth substrates, stress to, predominantly, specific inhibitors and drugs acting on various cellular processes. Furthermore, co-segregation of the targeted deletion and the observed phenotype(s) in meiotic products has been verified. The experimental procedure, using microtiter plates for phenotypic analysis of yeast mutants, can be applied on a large scale, either on solid or in liquid media. Since the minimal working unit of one 96-well microtiter plate allows the simultaneous analysis of at least 60 mutant strains, hundreds of strains can be handled in parallel. The high number of monotropic and pleiotropic phenotypes (62%) obtained, together with the acquired practical experience, have shown this approach to be simple, inexpensive and reproducible. It provides a useful tool for the yeast community for the systematic search of biochemical and physiological functions of unknown genes accounting for about a half of the 6000 genes of the complete yeast genome.

PetCR46, a gene which is essential for respiration and integrity of the mitochondrial genome.

In the frame of the European Pilot Project for the functional analysis of newly discovered open reading frames (ORFs) from Saccharomyces cerevisiae chromosome III, we have deleted entirely the YCR46C ORF by a one-step polymerase chain reaction method and replaced it by the HIS3 marker in the strain W303. The deletion has been checked by meiotic segregation and Southern blot analyses. Characterization of the deleted strain indicates that YCR46C is essential for respiration and maintenance of the mitochondrial genome since its deletion leads to the appearance of 100% of cytoplasmic petites. Hybridization with molecular probes from mtDNA of individual clones of such petites showed that about 50% did hybridize (rho- clones) while others did not (possibly rho degrees clones). The wild-type gene has been cloned and shown to complement the deletion. The gene, which probably codes for a mitochondrial ribosomal protein, has been called petCR46.

Structure-function relationships of the mitochondrial bc1 complex in temperature-sensitive mutants of the cytochrome b gene, impaired in the catalytic center N.

Seven new structures of cytochrome b have been recently identified by isolating and sequencing revertants from cytochrome b respiratory deficient mutants (Coppée, J. Y., Brasseur, G., Brivet-Chevillotte, P., and Colson, A. M. (1994) J. Biol. Chem. 269, 4221-4226). These mutations are located in the center N domain (QN). All the revertants exhibited a modified heme b562 maximum, confirming that part of the NH2-terminal region is in the vicinity of the extramembranous loop between helices IV-V and heme b562. Based on measurements performed on the maximal activities occurring in each segment of the respiratory chain, the decrease observed in the NADH oxidase activities of several revertants was correlated with some bc1 complex activity impairments; this may also explain why a moderate decrease in bc1 complex activity does not limit the succinate oxidase activity. The decrease in the rate of reduction of cytochrome b via the center N pathway is responsible for the impairment of the bc1 complex activity of these revertants. The three double-mutated revertants (S206L/N208K or -Y; S206L/W30C) are temperature-sensitive in vivo, and their mitochondria like that of the original mutant S206L are thermosensitive in vitro. Isolating the W30C mutation does not yield a thermosensitive phenotype: the replacement of serine 206 by leucine is therefore responsible for the thermoinstability of these strains; this temperature sensitivity is reinforced by additional mutations N208K or N208Y, and not by W30C. These data suggest that serine 206 and asparagine 208 are involved in the thermostability of the protein. When bc1 complex activity is lost after incubating mitochondria at a nonpermissive temperature (37 degrees C), heme b is still present, but can no longer be reduced by physiological substrate. The progressive loss of bc1 complex activity seems to be initially linked to a change in the tertiary structure of cytochrome b, which occurs drastically at center N and much more slowly at center P, as shown by kinetic study on the two cytochrome b redox pathways.

Genetic analysis and overexpression of lipolytic activity in Bacillus subtilis.

The previously cloned Bacillus subtilis lipase gene (lip) was mapped on the chromosome and used in the construction of a B. subtilis derivative totally devoid of any lip sequence. Homologous overexpression was performed in this strain by subcloning the lip open reading frame on a multicopy plasmid under the control of a strong gram-positive promoter. A 100-fold overproducing strain was obtained, which should facilitate purification of the secreted protein. Furthermore, the delta lip strain BCL1050 constitutes an ideal host for the cloning of heterologous lipase genes.

Non-native intragenic reversions selected from Saccharomyces cerevisiae cytochrome b-deficient mutants. Structural and functional features of the catalytic center N domain.

A total of 110 revertants have been isolated from two well-characterized cytochrome b deficient (mit-) mutants. The mit- mutations are located in an extramembranous loop linking the transmembrane alpha-helices IV and V of cytochrome b which has been postulated to be part of the catalytic center QN and therefore is assumed to be essential for the functioning of the bc1 complex. The molecular bases of the reversions were identified by sequencing the cytochrome b mRNAs. This allowed us to identify seven new structures of cytochrome b which are more or less compatible with its catalytic activity. The secondary mutations occurred either at the level of the original site mutation or at adjacent positions (region 204-208 of the polypeptide chain), or even at a distance of more than 150 amino acids (position 30) suggesting topological interaction between these two areas. All the revertants recovered cytochrome contents and phosphorylation efficiencies similar to the wild-type ones, albeit differences appeared in their specific growth rates and NADH respirations. The failure in bc1 complex functioning induced by the mutation S206L and its restoration by non native reversions are tentatively explained.

Analysis of revertants from respiratory deficient mutants within the center N of cytochrome b in Saccharomyces cerevisiae.

Four modified cytochrome b's carrying mononucleotide substitutions affecting center N residues were analysed. The mutant carrying a G33D change does not incorporate heme into the apocytochrome b and fails to grow on non-fermentable carbon sources. Out of 85 genetically independent revertants derived from this mutant, 82 were true back-mutants restoring the wild type sequence (D33G). The remaining three replaced the aspartic acid by an alanine (D33A) indicating that small size residues are best tolerated at this position which is consistent with the perfect conservation of the G33 during evolution. This glycine may be of crucial importance for helix packing around the hemes. The replacement of methionine at position 221 by lysine (M221K) produced a non-functional cytochrome b [(1993) J. Biol. Chem. 268, 15626-15632]. Non-native revertants replacing the lysine 221 by glutamic acid (K221E) or glutamine (K221Q) expressed a selective resistance to antimycin and antimycin derivatives having a modified dilactone ring moiety. Cytochrome b residues in 33 and in 221 seemed to contribute to the quinone reduction (QN) site of the cytochrome bc1 complex. Possible intramolecular interactions between the N-terminal region and the loop connecting helices IV and V of cytochrome b are proposed.

Molecular analysis of revertants from a respiratory-deficient mutant affecting the center o domain of cytochrome b in Saccharomyces cerevisiae.

In bc complexes, cytochrome b plays a major role in electron transfer and in proton translocation across the membrane. Several inhibitor-resistant and respiratory-deficient mutants have already been used to study the structure-function relationships of this integral membrane protein. We describe here the selection and the molecular analysis of revertants from a thermo-sensitive mit-mutant of known nucleotide changes. Among 80 independent pseudo-wild type revertants screened by DNA-labelled oligonucleotide hybridization, 33 have been sequenced. Eight suppressor mutations, affecting a region critical for both the function and the binding of center o inhibitors (end of helix C) were identified. Two of them were found to be more resistant to myxothiazol.

Molecular basis for resistance to myxothiazol, mucidin (strobilurin A), and stigmatellin. Cytochrome b inhibitors acting at the center o of the mitochondrial ubiquinol-cytochrome c reductase in Saccharomyces cerevisiae.

The respiratory bc1 complex transfers the electrons from ubiquinol to cytochrome c oxidase. Myxothiazol, strobilurin A (mucidin), and stigmatellin are center o inhibitors preventing electron transfer at the ubiquinone redox site Qo, which is located closer to the outer side of the inner mitochondrial membrane. The cytochrome b gene is carried by the organelle DNA. Yeast mutants resistant to myxothiazol and mucidin have been previously isolated and mapped to specific loci of the cytochrome b gene. In the present work, stigmatellin-resistant mutants were isolated and genetically analyzed. The mutated amino acid residues from seven myxothiazol-, four mucidin-, and six stigmatellin-resistant mutants have been identified by sequencing the relevant segments of the resistant cytochrome b gene. A third myxothiazol-resistant locus and the first stigmatellin-resistant locus were identified. The mutated codons were found to be clustered in two regions of the cytochrome b protein which appeared to be responsible for the resistance to Qo site inhibitors. The first region is within the end of the first, the second, and the beginning of the third exon whereas the second region is within exon five and the beginning of the sixth exon.