Bacterial swimming is mediated by rotation of a filament that is assembled via polymerization of flagellin monomers after secretion via a dedicated flagellar Type III secretion system. Several bacteria decorate their flagellin with sialic acid related sugars that is essential for motility. Aeromonas caviae is a model organism for this process as it contains a genetically simple glycosylation system and decorates its flagellin with pseudaminic acid (Pse). The link between flagellin glycosylation and export has yet to be fully determined. We examined the role of glycosylation in the export and assembly process in a strain lacking Maf1, a protein involved in the transfer of Pse onto flagellin at the later stages of the glycosylation pathway. Immunoblotting, established that glycosylation is not required for flagellin export but is essential for filament assembly since non-glycosylated flagellin is still secreted. Maf1 interacts directly with its flagellin substrate in vivo, even in the absence of pseudaminic acid. Flagellin glycosylation in a flagellin chaperone mutant (flaJ) indicated that glycosylation occurs in the cytoplasm before chaperone binding and protein secretion. Preferential chaperone binding to glycosylated flagellin revealed its crucial role, indicating that this system has evolved to favour secretion of the polymerization competent glycosylated form.

Bdellovibrio bacteriovorus is a small Gram-negative, obligate predatory bacterium that is largely found in wet, aerobic environments (e.g., soil). This bacterium attacks and invades other Gram-negative bacteria, including animal and plant pathogens. The intriguing life cycle of B. bacteriovorus consists of two phases: a free-living nonreplicative attack phase, in which the predatory bacterium searches for its prey, and a reproductive phase, in which B. bacteriovorus degrades a host’s macromolecules and reuses them for its own growth and chromosome replication. Although the cell biology of this predatory bacterium has gained considerable interest in recent years, we know almost nothing about the dynamics of its chromosome replication. Here, we performed a real-time investigation into the subcellular localization of the replisome(s) in single cells of B. bacteriovorus. Our results show that in B. bacteriovorus, chromosome replication takes place only during the reproductive phase and exhibits a novel spatiotemporal arrangement of replisomes. The replication process starts at the invasive pole of the predatory bacterium inside the prey cell and proceeds until several copies of the chromosome have been completely synthesized. Chromosome replication is not coincident with the predator cell division, and it terminates shortly before synchronous predator filament septation occurs. In addition, we demonstrate that if this B. bacteriovorus life cycle fails in some cells of Escherichia coli, they can instead use second prey cells to complete their life cycle. IMPORTANCE New strategies are needed to combat multidrug-resistant bacterial infections. Application of the predatory bacterium Bdellovibrio bacteriovorus, which kills other bacteria, including pathogens, is considered promising for combating bacterial infections. The B. bacteriovorus life cycle consists of two phases, a free-living, invasive attack phase and an intracellular reproductive phase, in which this predatory bacterium degrades the host’s macromolecules and reuses them for its own growth. To understand the use of B. bacteriovorus as a “living antibiotic,” it is first necessary to dissect its life cycle, including chromosome replication. Here, we present a real-time investigation into subcellular localization of chromosome replication in a single cell of B. bacteriovorus. This process initiates at the invasion pole of B. bacteriovorus and proceeds until several copies of the chromosome have been completely synthesized. Interestingly, we demonstrate that some cells of B. bacteriovorus require two prey cells sequentially to complete their life cycle.

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13Bdellovibrio bacteriovorus is a small Gram-negative, an obligate predatory bacterium that is 14 largely found in wet, aerobic environments (i.e. soil). This bacterium attacks and invades other 15 Gram-negative bacteria, including animal and plant pathogens. The intriguing life cycle of B. 16 bacteriovorus consists of two phases: a free-living non-replicative attack phase wherein the 17 predatory bacterium searches for its prey, and a reproductive phase, in which B. bacteriovorus 18 degrades a host's macromolecules and reuses them for its own growth and chromosome 19 replication. Although the cell biology of this predatory bacterium has gained considerable 20 interest in recent years, we know almost nothing about the dynamics of chromosome replication 21 in B. bacteriovorus. Here, we performed a real-time investigation into the subcellular 22 localization of the replisome(s) in single cells of B. bacteriovorus. Our results confirm that in 23 B. bacteriovorus chromosome replication fires only during the reproductive phase, and show 24 for the first time that this predatory bacterium exhibits a novel spatiotemporal arrangement of 25 chromosome replication. The replication process starts at the invasive pole of the predatory 26 bacterium inside the prey cell and proceeds until several copies of the chromosome have been 27 completely synthesized. This chromosome replication is not coincident with the predator-cell 28 division, and it terminates shortly before synchronous predator-filament septation occurs. In 29 addition, we demonstrate that if this lifecycle fails in some cells of B. bacteriovorus, they can 30 instead use two prey cells sequentially to complete their life cycle. 31 Importance 32 New strategies are needed to combat multidrug-resistant bacterial infections. Application of the 33 predatory bacterium, Bdellovibrio bacteriovorus, which kills other bacteria including 34 pathogens, is considered promising for bacterial infections. The B. bacteriovorus life cycle 35 consists of two phases, a free-living, invasive attack phase and an intracellular reproductive 36 phase, in which this predatory bacterium degrades the host's macromolecules and reuses them 37 3 for its own growth. To understand the use of B. bacteriovorus as a 'living antibiotic', it is first 38 necessary to dissect its life cycle including chromosome replication. Here, we present for the 39 first time a real-time investigation into subcellular localization of chromosome replication in a 40 single cells of B. bacteriovorus. This process initiates at the invasion pole of B. bacteriovorus 41 and proceeds until several copies of the chromosome have been completely synthesized. 42 Interestingly, we demonstrate that some cells of B. bacteriovorus require two prey cells 43 sequentially to complete their life cycle. 44 45Bdellovibrio bacteriovorus is a small (0.2-to 0.5-μm wide and 0.5-to 2.5-μm long) 46 Gram-negative bacterium that is unusual in its ability to invade and kill other Gram-negative 47 bacteria. Moreover, it was demonstrated th...