Summary paragraphBacteria have developed mechanisms to communicate and compete with one another in diverse environments 1. A new form of intercellular communication, contact-dependent growth inhibition (CDI), was discovered recently in Escherichia coli 2. CDI is mediated by the CdiB/CdiA two-partner secretion system. CdiB facilitates secretion of the CdiA ‘exoprotein’ onto the cell surface. An additional immunity protein (CdiI) protects CDI+ cells from autoinhibition 2, 3. The mechanisms by which CDI blocks cell growth and CdiI counteracts this growth arrest are unknown. Moreover, the existence of CDI activity in other bacteria has not been explored. Here we show that the CDI growth inhibitory activity resides within the carboxy-terminal region of CdiA (CdiA-CT), and that CdiI binds and inactivates cognate CdiA-CT, but not heterologous CdiA-CT. Bioinformatic and experimental analyses show that multiple bacterial species encode functional CDI systems with high sequence variability in the CdiA-CT and CdiI coding regions. CdiA-CT heterogeneity implies that a range of toxic activities are utilized during CDI. Indeed, CdiA-CTs from uropathogenic E. coli and the plant pathogen Dickeya dadantii have different nuclease activities, each providing a distinct mechanism of growth inhibition. Finally, we show that bacteria lacking the CdiA-CT and CdiI coding regions are unable to compete with isogenic wild-type CDI+ cells in both laboratory media and upon a eukaryotic host. Taken together, these results suggest that CDI systems constitute an intricate immunity network that plays an important role in bacterial competition.

Vaccination with the merozoite surface protein 3 (MSP3) of Plasmodium falciparum protects against infection in primates and is under development as a vaccine against malaria in humans. MSP3 is secreted and associates with the parasite membrane but lacks a predicted transmembrane domain or a glycosylphosphatidylinositol anchor. Its role in the invasion of red blood cells is unclear. To study MSP3, we produced recombinant full-length protein and found by size exclusion chromatography that the apparent size of MSP3 was much larger than predicted from its sequence. To investigate this, we used several biophysical techniques to characterize the fulllength molecule and four smaller polypeptides. The MSP3 polypeptides contain a large amount of ␣-helix and random coil secondary structure as measured by circular dichroism spectroscopy. The fulllength MSP3 forms highly elongated dimers and tetramers as revealed by chemical cross-linking and analytical ultracentrifugation. The dimer is formed through a leucine zipper-like domain located between residues 306 and 362 at the C terminus. Two dimers interact through their C termini to form a tetramer with an apparent association constant of 3 M. Sedimentation velocity experiments determined that the MSP3 molecules are highly extended in solution (some with f/f 0 > 2). These data, in light of the recent discoveries of three other Plasmodium proteins containing very similar C-terminal sequences, suggest that the members of this newly identified family may adopt highly extended and oligomeric novel structures capable of interacting with a red blood cell at relatively long distances.Plasmodium falciparum, the parasitic agent that causes most cases of fatal malaria, is estimated to infect over 500 million people annually (1), causing 1-3 million deaths among young children in sub-Saharan Africa. The rise of parasite resistance to currently available drugs has made malaria treatment difficult. Vaccines have great potential as lower cost alternatives to widespread drug treatment, since they may provide long lived protection from disease. The P. falciparum blood stage surface protein MSP3 2 (2, 3) is a vaccine candidate, since it is known to be a target of the immune response. Immunizations with several forms of recombinant MSP3 were each shown to protect monkeys against parasite challenge (4, 5). MSP3-specific antibodies in the presence of monocytes mediated killing of parasites in an antibody-dependent cellular inhibition assay (6). Homologs to MSP3 have been identified in several Plasmodium species (7-9), indicating that the molecule plays a conserved role in blood stage parasites. The P. falciparum MSP3 sequence predicts several domains ( Fig. 1): a central domain that includes three blocks of imperfect Ala heptad repeats of the sequence pattern Ala-X-X-Ala-X-X-X, a second central region rich in Glu residues, and a C-terminal leucine zipper-like domain (2, 3). Secondary structure algorithms predict MSP3 to be largely ␣-helical, especially in the Ala heptad repeat region that is pre...

Escherichia coli transcription termination factor Rho is a ring-shaped hexameric protein that uses the energy derived from ATP hydrolysis to dissociate RNA transcripts from the ternary elongation complex. To test a current model for the interaction of Rho with RNA, three derivatives of Rho were made containing single cysteine residues and modified with a photo-activable cross-linker. The positions for the cysteines were: 1) in part of the primary RNA-binding site in the N terminus (Cys-82 Rho); 2) in a connecting polypeptide proposed to be on the outside of the hexamer (Cys-153 Rho); and 3) near the proposed secondary RNA-binding site in the ATP-binding domain (Cys-325 Rho). Results from the cross-linking of the modified Rho proteins to a series of cro RNA derivatives showed that Cys-82 Rho formed cross-links with all transcripts containing the Rho utilization (rut) site, that Cys-325 Rho formed cross-links to transcripts that had the rut site and 10 or more residues 3 of the rut site, and that Cys-153 did not form crosslinks with any of the transcripts. From a model of the quaternary structure of Rho, which is largely based on homology to the F 1 -ATPase, amino acid 82 is located near the top of the hexamer, and amino acid 325 is located on a solvent-accessible loop in the center of the hexamer. These data are consistent with binding of the rut region of RNA around the crown, with its 3 -segment passing through the center of the Rho hexamer.Transcription termination factor Rho in Escherichia coli consists of six identical protein subunits arranged in a ring structure (1, 2). For its function in termination, Rho binds to the nascent transcript and acts to dissociate the transcript from RNA polymerase and the DNA template (3, 4). Rho can also act as a helicase to dissociate a DNA molecule that is base paired to a 3Ј-segment of an RNA with a Rho attachment site (5, 6). The motive power for these actions comes from the hydrolysis of ATP to ADP and P i . The mechanism for coupling ATP hydrolysis to these dissociation reactions is not known.The Rho polypeptide contains two major domains: an Nterminal RNA-binding domain (residues 1-130) and a C-terminal ATP-binding domain (residues 131-419) (7,8). The RNAbinding domain contains an oligosaccharide/oligonucleotidebinding domain (OB-fold) (9). Rho forms strong binding interactions with single-stranded C-rich RNA molecules, and RNA segments with these characteristics form the attachment sites (known as the rut or Rho utilization sites) used by Rho to mediate termination of a transcript. The structure of a complex of the RNA-binding domain of Rho with oligo(rC) 9 , determined by x-ray crystallography, shows that a cytidylate residue forms H bonds with Arg-66 and Glu-78 and a -bond stacking interaction with Phe-64 (10).Evidence for a second RNA-binding site distinct from that in the RNA-binding domain has come from a detailed analysis of the polynucleotide requirements for activation of ATP hydrolysis by Rho (11) and from the finding that mutant forms of Rho with changes in...

Escherichia coli Rho factor is a ring-shaped, homohexameric protein that terminates synthesis of RNA through interactions with the nascent RNA transcript. Because its mechanism of action may involve translocation of the RNA transcript through the hole in its ring structure, its action could depend on the availability of a free 5 terminus. To determine whether Rho's activity is 5-end-dependent, its ability to bind to and function on a circular derivative of cro mRNA was investigated. The circular derivative was made in vitro by action of RNA ligase on a derivative of cro RNA containing an extra 10-nucleotide sequence near the 5-end that was complementary to a sequence located near the 3-end. Rho bound nearly as tightly to the circular derivative RNA as to the standard cro transcript. Rho was also able to readily dissociate a DNA oligonucleotide from its helical complex with the circular RNA in an ATP-dependent reaction. Thus, the action of Rho on a transcript does not depend on the availability of a free 5 terminus.Transcription termination factor Rho is an RNA-binding protein that couples the energy derived from ATP hydrolysis to actions that dissociate a RNA transcript from its biosynthetic complex with DNA and RNA polymerase (1, 2). Rho also can dissociate a RNA molecule bound to DNA through a short hybrid helix in a reaction that is dependent on ATP hydrolysis and the presence of an attachment site for Rho on the RNA on the 5Ј side of the hybrid helix (3-5). This RNA-DNA helicase activity may mimic the process of removal of the nascent RNA strand from the transcription elongation complex.Rho factor is believed to function in vivo as a hexamer consisting of six polypeptide subunits arranged in a ring-shaped structure (6, 7). Burgess and Richardson (8) have recently presented a model for the Rho-RNA complex in which a segment of RNA, called a rut site (Rho utilization site), binds to a cleft comprised of the N-terminal RNA-binding domains of the six individual subunits located at one end of the hexameric structure (the crown) (9 -11). RNA sequence 3Ј of the rut site then passes into the hole located at the center of the hexamer. Evidence for passage of a single-stranded nucleic acid substrate through a ring-shaped hexameric structure has been observed for other hexameric helicases, such as DnaB and the T7 gene 4 product (12, 13).The process by which Rho binds to and captures the 3Ј-end of an RNA in the center of the hexamer is not known. Gan and Richardson (14) have recently presented data indicating that Rho, in vitro, could form hexamers by partial assembly of subunits on an RNA. Another mechanism could have the RNA enter into the center of the hexameric structure through an opening or notch in the ring (7). A third mechanism may involve Rho threading onto the nascent transcript from the free 5Ј-end of the RNA. To test the model in which Rho requires a free end of RNA to thread onto in order to function as a terminator, we investigated Rho's ability to utilize its ATP-dependent helicase activity on an RNA lac...