Summary Bacterial secretion systems often employ molecular chaperones to recognize and facilitate export of their substrates. Recent work demonstrated that a secreted component of the type VI secretion system (T6SS), hemolysin co-regulated protein (Hcp), binds directly to effectors, enhancing their stability in the bacterial cytoplasm. Herein, we describe a quantitative cellular proteomics screen for T6S substrates that exploits this chaperone-like quality of Hcp. Application of this approach to the Hcp secretion island I-encoded T6SS (H1-T6SS) of Pseudomonas aeruginosa led to the identification of a novel effector protein, termed Tse4 (type VI secretion exported 4), subsequently shown to act as a potent intra-specific H1-T6SS-delivered antibacterial toxin. Interestingly, our screen failed to identify two predicted H1-T6SS effectors, Tse5 and Tse6, which differ from Hcp-stabilized substrates by the presence of toxin-associated PAAR-repeat motifs and genetic linkage to members of the valine-glycine repeat protein G (vgrG) genes. Genetic studies further distinguished these two groups of effectors: Hcp-stabilized effectors were found to display redundancy in interbacterial competition with respect to the requirement for the two H1-T6SS-exported VgrG proteins, whereas Tse5 and Tse6 delivery strictly required a cognate VgrG. Together, we propose that interaction with either VgrG or Hcp defines distinct pathways for T6S effector export.

Bacterial contact-dependent growth inhibition (CDI) is mediated by the CdiB/CdiA family of two-partner secretion proteins. CdiA effector proteins are exported onto the surface of CDI+ inhibitor cells, where they interact with susceptible bacteria and deliver effectors/toxins derived from their C-terminal regions (CdiA-CT). CDI+ cells also produce an immunity protein that binds the CdiA-CT and blocks its activity to prevent autoinhibition. Here, we show that the CdiA-CT from uropathogenic Escherichia coli strain 536 (UPEC536) is a latent tRNase that requires activation by the biosynthetic enzyme CysK (O-acetylserine sulfhydrylase A). UPEC536 CdiA-CT exhibits no nuclease activity in vitro, but cleaves within transfer RNA (tRNA) anti-codon loops when purified CysK is added. CysK and CdiA-CT form a stable complex, and their binding interaction appears to mimic that of the CysK/CysE cysteine synthase complex. CdiA-CT activation is also required for growth inhibition. Synthesis of CdiA-CT in E. coli cysK+ cells arrests cell growth, whereas the growth of ΔcysK mutants is unaffected by the toxin. Moreover, E. coli ΔcysK cells are completely resistant to inhibitor cells expressing UPEC536 CdiA, indicating that CysK is required to activate the tRNase during CDI. Thus, CysK acts as a permissive factor for CDI, providing a potential mechanism to modulate growth inhibition in target cells.

Summary Contact-dependent growth inhibition (CDI) is one mechanism of inter-bacterial competition. CDI+ cells export large CdiA effector proteins, which carry a variety of C-terminal toxin domains (CdiA-CT). CdiA-CT toxins are specifically neutralized by cognate CdiI immunity proteins to protect toxin-producing cells from auto-inhibition. Here, we use structure determination to elucidate the activity of a unique CDI toxin from Enterobacter cloacae (ECL). The structure of CdiA-CTECL resembles the C-terminal nuclease domain of colicin E3, which cleaves 16S ribosomal RNA to disrupt protein synthesis. In accord with this structural homology, we show that CdiA-CTECL uses the same nuclease activity to inhibit bacterial growth. Surprisingly, although colicin E3 and CdiAECL carry equivalent toxin domains, the corresponding immunity proteins are unrelated in sequence, structure and toxin-binding site. Together, these findings reveal unexpected diversity amongst 16S rRNases and suggest that these nucleases are robust and versatile payloads for a variety of toxin-delivery platforms.

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Abstract. This study was undertaken to prove that voltage-sensitive calcium channels controlling the photophobic stop response of the unicellular green alga Chlamydomonas reinhardtii are exclusively found in the flagellar region of the cell and to answer the question as to their exact localization within the flagellar membrane. The strategy used was to amputate flagella to a variable degree without perturbing the electrical properties of the cell and measure flagellar currents shortly after amputation and during the subsequent regeneration process. Under all conditions, a close correlation was found between current size and flagellar length, strongly suggesting that the channels that mediate increases in intraflagellar calcium concentration are confined to and distributed over the total flagellar length. Bald mutants yielded tiny flagellar currents, in agreement with the existence of residual flagellar stubs. In the presence of the protein synthesis inhibitor cycloheximide, flagellar length and flagellar currents also recovered in parallel. Recovery came to an earlier end, however, leveling off at a time when in the absence of cycloheximide only half maximal values were achieved. This suggests the existence of a pool of precursors, which permits the maintenance of a constant ratio between voltage-sensitive calcium channels and other intraflagellar proteins. CHLAMYDOMONAS reinhardtii, a unicellular green freshwater alga, possesses a photosensory system and two flagella, which enable it to translate information about intensity and direction of the ambient light into defined orientational responses (Boscov and Feinleib, 1979). During normal forward swimming the two flagella move in a breast stroke-like fashion 50 times per second. An apparent asymmetry in the beating pattern leads to a rotation around the longitudinal axis with a frequency of 2 Hz. Thereby, one revolution takes between 20 and 25 strokes, i.e., 500 ms. This allows the alga to screen the ambient light and to correct for changes in its direction (Foster and Smyth, 1980;Riiffer and Nultsch, 1985, 1990. Depending on the brightness of the actinic light, three classes of behavioral responses can be distinguished: (a) in weak light, Chlamydomonas swims towards the light (positive phototaxis); (b) in stronger light, it swims away from it (negative phototaxis); and (c) in very strong light, particularly when there are sudden and pronounced changes in intensity, it stops transiently. In the course of this "stop response; Chlamydomonas changes from its normal forward swimming to a phase of slower backward swimming, caused by an undulation movement of the flagella. Afterwards it resumes forward swimming in a new direction. Stop responses start within 50 ms of the stimulus and last for about 300 ms (Uhl and Hegemann, 1990).The authors' present address is Botanisches Institut der Ludwig Maximilians Universitit, Menzinger Strasse 67, D-80638 Miinchen, Germany.There is good evidence that the different flageUar beating patterns are all governed by the internal calci...