The Rcs phosphorelay signal transduction system controls genes for capsule production and many other envelope-related functions and is implicated in biofilm formation. We investigated the activation of the Rcs system in a pgsA null mutant of Escherichia coli, which completely lacks the major acidic phospholipids phosphatidylglycerol and cardiolipin. We found that the Rcs activation, and consequent thermosensitivity, were suppressed by overexpression of the lgt gene, encoding diacylglyceryltransferase, which catalyses the modification of prolipoproteins that is the first step in the maturation and localization process of lipoproteins, and is a prerequisite for the later steps. The outer-membrane lipoprotein RcsF is an essential component of Rcs signalling. This lipoprotein was poorly localized to the outer membrane in the pgsA null mutant, probably because of the absence of phosphatidylglycerol, the major donor of diacylglycerol in the Lgt reaction. Even in a pgsA + background, the Rcs system was activated when RcsF was mislocalized to the inner membrane by alteration of the residues at positions 2 and 3 of its mature form to inner-membrane retention signals, or when it was mislocalized to the periplasm by fusing the mature form to maltose-binding protein. These results suggest that RcsF functions as a ligand for RcsC in activating Rcs signalling. Mislocalized versions of RcsF still responded to mutations pgsA, mdoH and tolB, further activating the Rcs system, although the rfaP mutation barely caused activation. It seems that RcsF must be localized in the outer membrane to respond effectively to stimuli from outside the cell.

Abstract:We developed a novel protocol with superior quantitative analysis results for DNA metabarcoding of Collembola, a major soil microarthropod order. Degenerate PCR primers were designed for conserved regions in the mitochondrial cytochrome c oxidase subunit I (mtCOI) and 16S ribosomal RNA (mt16S) genes based on published collembolan mitogenomes. The best primer pair was selected based on its ability to amplify each gene, irrespective of the species. DNA was extracted from 10 natural communities sampled in a temperate forest (with typically 25-30 collembolan species per 10 soil samples) and 10 mock communities (with seven cultured collembolan species). The two gene regions were then amplified using the selected primers, ligated with adapters for 454 technology, and sequenced. Examination of the natural community samples showed that 32 and 36 operational taxonomic units (defined at a 90% sequence similarity threshold) were recovered from the mtCOI and mt16S data, respectively, which were comparable to the results of the microscopic identification of 25 morphospecies. Further, sequence abundances for each collembolan species from the mtCOI and mt16S data of the mock communities, after normalization by using a species as the internal control, showed good correlation with the number of individuals in the samples (R = 0.91-0.99), although relative species abundances within a mock community sample estimated from sequences were skewed from community composition in terms of the number of individuals or biomass of the species. Thus, this protocol enables the comparison of collembolan communities in a quantitative manner by metabarcoding.Key words: metabarcoding, Collembola, 16S, COX1, quantification. Résumé :Les auteurs ont mis au point un protocole pour le métacodage à barres de l'ADN chez les collemboles, un ordre important parmi les micro-arthropodes du sol. Des amorces PCR dégénérées ont été conçues pour les gènes codant pour la sous-unité I de la cytochrome c oxydase mitochondriale (mtCOI) et pour l'ARN ribosomique 16S mitochondrial (mt16S) sur la base des génomes mitochondriaux déjà séquencés chez les collemboles. La meilleure paire d'amorces a été choisie sur la base de sa capacité à amplifier chaque gène, sans égard à l'espèce.

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Escherichia coli pgsA mutations, which cause acidic phospholipid deficiency, repress transcription of the flagellar master operon flhDC, and thus impair flagellar formation and motility. The molecular mechanism of the strong repression of flhDC transcription in the mutant cells, however, has not yet been clarified. In order to shed light on this mechanism we isolated genes which, when supplied in multicopy, suppress the repression of flhD, and found that three genes, gadW, metE and yeaB, were capable of suppression. Taking into account a previous report that gadW represses s S production, the level of s S in the pgsA3 mutant was examined. We found that pgsA3 cells had a high level of s S and that introduction of a gadW plasmid into pgsA3 cells did reduce the s S level. The pgsA3 cells exhibited a sharp increase in s S levels that can only be partially attributed to the slight increase in rpoS transcription; the largest part of the effect is due to a post-transcriptional accumulation of s S . GadW in multicopy exerts its effect by posttranscriptionally downregulating s S . YeaB and MetE in multicopy also exert their effect via s S .Disruption of rpoS caused an increase of the flhD mRNA level, and induction from P trc -rpoS repressed the flhD mRNA level. The strong repression of flhD transcription in pgsA3 mutant cells is thus suggested to be caused by the accumulated s S . INTRODUCTIONEscherichia coli membranes contain only three major phospholipids. Molecular genetic approaches to correlate mutationally modified lipid compositions with altered membrane functions have been successfully used to understand the biological roles of the individual phospholipids (for reviews, see Shibuya, 1992;Cronan, 2003;Dowhan et al., 2004). Lack of phosphatidylethanolamine, a zwitterionic phospholipid, which accounts for about 70 % of total phospholipids, is lethal, but the lethality is suppressed by supplementation with high concentrations of divalent cations (DeChavigny et al., 1991;Saha et al., 1996). The lack of phosphatidylethanolamine causes a defect in motility (Shi et al., 1993), an activation of the Cpx two-component signal transduction pathway (Mileykovskaya & Dowhan, 1997), a defect in division site selection, probably due to the concomitant increase in cardiolipin content that affects the localization of MinD (Mileykovskaya & Dowhan, 2005), and a defect in the native folding of lactose permease LacY, which causes the loss of active transport of the substrate (Dowhan et al., 2004).Lack of, or deficiency in, the major acidic phospholipid phosphatidylglycerol, brought about by the mutations pgsA30 : : kan or pgsA3, respectively, is lethal, due to the absence of, or the missense defect in, phosphatidylglycerophosphate synthase, which catalyses the committed step in the biosynthetic pathway for this acidic phospholipid (Shibuya, 1992;Usui et al., 1994). However, simultaneous lack of the major outer membrane lipoprotein (Braun's lipoprotein), the most abundant protein, encoded by lpp, suppresses the lethality (Shibuya, 1992;K...