Downregulation of the motA gene delays the escape of the obligate predator Bdellovibrio bacteriovorus 109J from bdelloplasts of bacterial prey cells

Abstract

Bdellovibrio bacteriovorus is a Gram-negative bacterium that preys on other Gram-negative bacteria. The lifecycle of B. bacteriovorus alternates between an extracellular flagellated and highly motile non-replicative attack-phase cell and a periplasmic non-flagellated growth-phase cell. The prey bacterium containing periplasmic bdellovibrios becomes spherical but osmotically stable, forming a structure known as the bdelloplast. After completing the growth phase, newly formed bdellovibrios regain their flagellum and escape the bdelloplast into the environment, where they encounter more prey bacteria. The obligate predatory nature of B. bacteriovorus imposes a major difficulty to introducing mutations in genes directly involved in predation, since these mutants could be non-viable. This work reports the cloning of the B. bacteriovorus 109J motAB operon, encoding proteins from the flagellar motor complex, and a genetic approach based on the expression of a motA antisense RNA fragment to downregulate motility. Periplasmic bdellovibrios carrying the plasmid expressing antisense RNA displayed a marked delay in escaping from bdelloplasts, while the released attack-phase cells showed altered motility. These observations suggest that a functionally intact flagellar motor is required for the predatory lifecycle of B. bacteriovorus. Also, the use of antisense RNA expression may be a useful genetic tool to study the Bdellovibrio developmental cycle.

Three motAB Stator Gene Products in Bdellovibrio bacteriovorus Contribute to Motility of a Single Flagellum during Predatory and Prey-Independent Growth

“…Prey entry and flagellum-mediated movement within bdelloplasts. As we had found that contrary to the results of Flannagan and coworkers, (8), the B. bacteriovorus motAB1 strain was not significantly slower at predation during multiple rounds of infection, a single round of infection by each of the motAB1 and motAB2 strains was compared to that by the fliC1 merodiploid control by light microscopy in a "bdelloplast persistence assay." Analysis of these single rounds of infection (see Fig.…”

“…In the initial annotation of each motA gene (21), they were found to encode either an extended N-terminal sequence (motA1/motA2) or a missing first transmembrane domain (motA3), but further sequence analysis revealed alternative potential start codons that rectified these issues and gave significant full-length homologies to Mot proteins of diverse other bacteria. As mentioned above MotA1 of B. bacteriovorus HD100 was a 100% identical match to the MotA of B. bacteriovorus 109J studied with antisense knockdown by Flannagan and coworkers (8). We examined the primary sequence similarities between the six Bdellovibrio MotA and MotB protein sequences in comparison to MotA and MotB from the proton-driven E. coli motor and PomA and PomB from the Vibrio alginolyticus sodium-driven motor to see if any obvious homologies suggesting a driving ion for each MotAB type could be determined.…”

“…As Flannagan et al (8) reported that downregulation of a motA1 homologue in B. bacteriovorus 109J resulted in significantly diminished predation due to delayed escape from bdelloplasts, causing persistence of the downregulated motA1 strain inside bdelloplasts for 30 h or more, we studied motility of and predation by HD mutant strains. Motility analysis with a Hobson BacTracker (Table 2) showed that deletion of motAB1 or motAB2 resulted in reduced percent motility for the culture, but not reduced swimming speeds compared to wild-type levels, but that deletion of motAB3 resulted in significant reductions in both percent motility (from 98% for the wild-type fliC1 merodiploid to 25% for the motAB3 mutant) and swimming speed (from 63 m/s for the wild type to 27 m/s for the motAB3 mutant).…”

“…The Bd0144 motA gene of B. bacteriovorus HD100 showed 100% identity, at DNA and protein levels, to the motA gene in B. bacteriovorus 109J described by Flannagan et al (8), and there was also synteny with its surrounding genetic locus, including motB, between the two strains.…”

“…It then begins to digest the prey cell cytoplasm, using the broken-down contents to grow into an elongated growth-phase cell which, upon exhaustion of the prey cell cytoplasmic contents, septates into multiple progeny. The progeny then become flagellate, and there are previous reports (8,28) of flagellum-mediated motility within the remnants of the bdelloplast immediately prior to lysis of the prey outer membrane. After the bdelloplast is lysed, the progeny Bdellovibrio bacteria are released as highly motile attack-phase cells, seeking more prey.…”