No abstract

We report the identification of the proteins encoded by the mttABC operon (formerly yigTUW), which mediate a novel Sec-independent membrane targeting and translocation system in Escherichia coli that interacts with cofactor-containing redox proteins having a S/TRRXFLK "twin arginine" leader motif. A pleiotropic-negative mutant in mttA prevents the periplasmic localization of twin arginine redox enzymes, including nitrate reductase (NapA) and trimethylamine N-oxide reductase (TorA). The mutation also prevents the correct localization of the integral membrane molybdoenzyme dimethylsulfoxide reductase (DmsABC). The DmsA subunit has a twin arginine leader. Proteins with a Sec-dependent leader or which assemble spontaneously in the membrane are not affected by this mutation. MttA, B, and C are members of a large family of related sequences extending from archaebacteria to higher eukaryotes.

Existing high-throughput methods to identify RNA-binding proteins (RBPs) involving capture of polyadenylated RNAs can not recover proteins that interact with non-adenylated RNAs, including lncRNA, pre-mRNA and bacterial RNAs. We present orthogonal organic phase separation (OOPS) which does not require molecular tagging or capture of polyadenylated RNA. We verify OOPS in HEK293, U2OS and MCF10A human cell lines, finding 96% of proteins recovered are bound to RNA. We demonstrate that all long RNAs can be crosslinked to proteins and recover 1838 RBPs, including 926 putative novel RBPs. Importantly, OOPS is approximately 100-fold more efficient than current techniques, enabling analysis of dynamic RNA-protein interactions. We identified 749 proteins with altered RNA binding following release from nocodazole arrest. Finally, OOPS allowed the characterisation of the first RNA-interactome for a bacterium, Escherichia coli. OOPS is an easy to use and flexible technique, compatible with downstream proteomics and RNA sequencing and applicable to any organism.

The Amt proteins are ammonium transporters that are conserved throughout all domains of life, being found in bacteria, archaea and eukarya. In bacteria and archaea, the Amt structural genes (amtB) are invariably linked to glnK, which encodes a member of the P II signal transduction protein family, proteins that regulate enzyme activity and gene expression in response to the intracellular nitrogen status. We have now shown that in Escherichia coli and Azotobacter vinelandii, GlnK binds to the membrane in an AmtBdependent manner and that GlnK acts as a negative regulator of the transport activity of AmtB. Membrane binding is dependent on the uridylylation state of GlnK and is modulated according to the cellular nitrogen status such that it is maximal in nitrogen-suf®cient situations. The membrane sequestration of GlnK by AmtB represents a novel form of signal transduction in which an integral membrane transport protein functions to link the extracellular ammonium concentration to the intracellular responses to nitrogen status. The results also offer new insights into the evolution of P II proteins and a rationale for their trigonal symmetry.

Abstracti mb_942 249..258The pea aphid genome includes 66 genes contributing to amino acid biosynthesis and 93 genes to amino acid degradation. In several respects, the pea aphid gene inventory complements that of its symbiotic bacterium, Buchnera aphidicola (Buchnera APS). Unlike other insects with completely sequenced genomes, the pea aphid lacks the capacity to synthesize arginine, which is produced by Buchnera APS. However, consistent with other insects, it has genes coding for individual reactions in essential amino acid biosynthesis, including threonine dehydratase and branched-chain amino acid aminotransferase, which are not coded in the Buchnera APS genome. Overall the genome data suggest that the biosynthesis of certain essential amino acids is shared between the pea aphid and Buchnera APS, providing the opportunity for precise aphid control over Buchnera metabolism.