Pathogenic mycobacteria resist lysosomal delivery after uptake into macrophages, allowing them to survive intracellularly. We found that the eukaryotic-like serine/threonine protein kinase G from pathogenic mycobacteria was secreted within macrophage phagosomes, inhibiting phagosome-lysosome fusion and mediating intracellular survival of mycobacteria. Inactivation of protein kinase G by gene disruption or chemical inhibition resulted in lysosomal localization and mycobacterial cell death in infected macrophages. Besides identifying a target for the control of mycobacterial infections, these findings suggest that pathogenic mycobacteria have evolved eukaryotic-like signal transduction mechanisms capable of modulating host cell trafficking pathways.

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While in most rod-shaped bacteria, morphology is based on MreB-like proteins that form an actin-like cytoskeletal scaffold for cell wall biosynthesis, the factors that determine the more flexible rod-like shape in actinobacteria such as Mycobacterium species are unknown. Here we show that a Mycobacterium smegmatis protein homologous to eubacterial DivIVA-like proteins, including M. tuberculosis antigen 84 (Ag84), localized symmetrically to centers of peptidoglycan biosynthesis at the poles and septa. Controlled gene disruption experiments indicated that the gene encoding Ag84, wag31, was essential; when overexpressed, cells became longer and wider, with Ag84 asymmetrically distributed at one pole. Many became grossly enlarged, bowlingpin-shaped cells having up to 80-fold-increased volume. In these cells, Ag84 accumulated predominantly at a bulbous pole that was apparently generated by uncontrolled cell wall expansion. In some cells, Ag84 was associated with exceptional sites of cell wall expansion (buds) that evolved into branches. M. bovis BCG Ag84 was able to form oligomers in vitro, perhaps reflecting its superstructure in vivo. These data suggested a role for Ag84 in cell division and modulating cell shape in pleiomorphic actinobacteria.

SummaryThe Tol/Pal system of Escherichia coli is composed of the YbgC, TolQ, TolA, TolR, TolB, Pal and YbgF proteins. It is involved in maintaining the integrity of the outer membrane, and is required for the uptake of group A colicins and DNA of filamentous bacteriophages. To identify new interactions between the components of the Tol/Pal system and gain insight into the mechanism of colicin import, we performed a yeast two-hybrid screen using the different components of the Tol/Pal system and colicin A. Using this system, we confirmed the already known interactions and identified several new interactions. TolB dimerizes and the periplasmic domain of TolA interacts with YbgF and TolB. Our results indicate that the central domain of TolA (TolAII) is sufficient to interact with YbgF, that the C-terminal domain of TolA (TolAIII) is sufficient to interact with TolB, and that the amino terminal domain of TolB (D1) is sufficient to bind TolAIII. The TolA/TolB interaction was confirmed by crosslinking experiments on purified proteins. Moreover, we show that the interaction between TolA and TolB is required for the uptake of colicin A and for the membrane integrity. These results demonstrate that the TolA/TolB interaction allows the formation of a trans-envelope complex that brings the inner and outer membranes in close proximity. IntroductionThe Tol/Pal system is a multiprotein complex embedded into the envelope of Escherichia coli. The proteins of this system are encoded by a cluster of genes organized into two operons. The first one encodes YbgC, TolQ, TolR, TolA, TolB, Pal and YbgF and the second one from an system might catalyse porin biogenesis or regulate porin activity as TolB and TolA interact with porins (Dérouiche et al., 1996;Rigal et al., 1997;Dover et al., 2000). Finally, this system might drive macromolecules through the envelope as it has been recently suggested that TolQ-TolR could function as a motor energizing TolA (Cascales et al., 2000;2001;Gaspar et al., 2000;Germon et al., 2001).The organization of the Tol/Pal system in the envelope has been extensively studied. Several experiments, such as cross-linking with formaldehyde, coimmunoprecipitations and genetic suppression experiments, have shown that these proteins interact with each other and are organized into two complexes. The inner membrane complex contains TolA, TolQ and TolR. The TolA transmembrane domain interacts with the first transmembrane domain of TolQ and with TolR transmembrane domain Germon et al., 1998). The first and the third transmembrane domains of TolQ seem to be in close contact . The third transmembrane domain of TolQ interacts with TolR transmembrane domain and with TolR dimer. TolRII and TolRIII, the two periplasmic domains of TolR, have the intrinsic capacity to dimerize (Journet et al., 1999). The second complex, associated to the outer membrane, is composed of TolB, Pal, Lpp and OmpA. TolB interacts with Pal, but also with Lpp and OmpA (Bouveret et al., 1995;Clavel et al., 1998;Ray et al., 2000). The only direct evidence...