SummaryBacteria of Shigella spp. are responsible for shigellosis in humans and use a type III secretion (TTS) system to enter epithelial cells and trigger apoptosis in macrophages. Transit of translocator and effector proteins through the TTS apparatus is activated upon contact of bacteria with host cells. Transcription of ª ª ª ª 15 genes encoding effectors is regulated by the TTS apparatus activity and controlled by MxiE, an AraC family activator, and its coactivator IpgC, the chaperone of IpaB and IpaC translocators. Using a genetic screen, we identified ospD1 as a gene whose product negatively controls expression of genes regulated by secretion activity. OspD1 associates with the chaperone Spa15 and the activator MxiE and acts as an antiactivator until it is secreted. The mechanism regulating transcription in response to secretion activity involves an activator (MxiE), an anti-activator (OspD1), a co-anti-activator (Spa15), a coactivator (IpgC) and two anti-coactivators (IpaB and IpaC) whose alternative and mutually exclusive interactions are controlled by the duration of the TTS apparatus activity.

In Lactococcus lactis, cell-wall polysaccharides (CWPSs) act as receptors for many bacteriophages, and their structural diversity among strains explains, at least partially, the narrow host range of these viral predators. Previous studies have reported that lactococcal CWPS consists of two distinct components, a variable chain exposed at the bacterial surface, named polysaccharide pellicle (PSP), and a more conserved rhamnan chain anchored to, and embedded inside, peptidoglycan. These two chains appear to be covalently linked to form a large heteropolysaccharide. The molecular machinery for biosynthesis of both components is encoded by a large gene cluster, named cwps. In this study, using a CRISPR/Cas-based method, we performed a mutational analysis of the cwps genes. MALDI-TOF MS-based structural analysis of the mutant CWPS combined with sequence homology, transmission EM, and phage sensitivity analyses enabled us to infer a role for each protein encoded by the cwps cluster. We propose a comprehensive CWPS biosynthesis scheme in which the rhamnan and PSP chains are independently synthesized from two distinct lipid-sugar precursors and are joined at the extracellular side of the cytoplasmic membrane by a mechanism involving a membrane-embedded glycosyltransferase with a GT-C fold. The proposed scheme encompasses a system that allows extracytoplasmic modification of rhamnan by complex substituting oligo-/polysaccharides. It accounts for the extensive diversity of CWPS structures observed among lactococci and may also have relevance to the biosynthesis of complex rhamnose-containing CWPSs in other Gram-positive bacteria.

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