Identification of Genes Essential for Prey-Independent Growth of Bdellovibrio bacteriovorus HD100

Abstract

Bdellovibrio bacteriovorus HD100 is an obligate predatory bacterium that attacks and invades Gram-negative bacteria. The predator requires living bacteria to survive as growth and replication take place inside the bacterial prey. It is possible to isolate mutants that grow and replicate outside prey bacteria. Such mutants are designated host or prey independent, and their nutritional requirements vary. Some mutants are saprophytic and require prey extracts for extracellular growth, whereas other mutants grow axenically, which denotes the formation of colonies on complete medium in the absence of any prey components. The initial events leading to prey-independent growth are still under debate, and several genes may be involved. We selected new mutants by three different methods: spontaneous mutation, transposon mutagenesis, and targeted gene knockout. By all approaches we isolated mutants of the hit (host interaction) locus. As the relevance of this locus for the development of prey independence has been questioned, we performed whole-genome sequencing of five prey-independent mutants. Three mutants were saprophytic, and two mutants could grow axenically. Wholegenome analysis revealed that the mutation of a small open reading frame of the hit locus is sufficient for the conversion from predatory to saprophytic growth. Complementation experiments were performed by introduction of a plasmid carrying the wild-type hit gene into saprophytic mutants, and predatory growth could be restored. Whole-genome sequencing of two axenic mutants demonstrated that in addition to the hit mutation the colony formation on complete medium was shown to be influenced by the mutations of two genes involved in RNA processing. Complementation experiments with a wild-type gene encoding an RNA helicase, RhlB, abolished the ability to form colonies on complete medium, indicating that stability of RNA influences axenic growth.

Cell-cycle progress in obligate predatory bacteria is dependent upon sequential sensing of prey recognition and prey quality cues

“…3A). Furthermore, a predator mutated in pcnB, a mutation that enables type I HI mutants to become completely axenic (37), still required the presence of ghosts for growth (see below and Fig. 5).…”

“…Type I HI mutants arise at low frequency (10 −7 ) and can grow saprophytically in the presence of prey extract (32,37). To confirm that prey extract-dependent ex vivo growth did not result from the selection of HI mutants but was still seen in WT cells, DNA was purified from AP cells and from ex vivo-cultivated GP cells.…”

“…4). Finally, earlier work had shown that deletion of pcnB, encoding for poly(A) polymerase, alleviates growth dependency on prey extract in type II HI mutants (37). We hypothesized that a ΔpcnB mutant in an otherwise WT background would create a strain still dependent on the presence of ghosts but independent of prey extract.…”

“…Mutations in gene bd0108 yield type I HI mutants that grow in the absence of prey in a rich medium supplemented with prey extract, i.e., they require the second cue (32,36,37). Type II HI mutants eliminate the requirement for prey extract; they are generated by a mutation in rhlB or in pcnB, genes that encode for RNA degradosome subunits (37). Although useful in research, HI mutants exhibit deregulated transitions between phases, as demonstrated by alterations in gene and protein expression (38,39).…”

The Bdellovibrio bacteriovorus twin-arginine transport system has roles in predatory and prey-independent growth

“…Two genes, Bd1802 and Bd3906, encoding potential Tat substrates were knocked out by silent deletion using modifications of the techniques used by Steyert & Pineiro (2007) and Roschanski et al (2011). Approximately 450 bp of flanking DNA from either side of the gene was amplified using primers listed in Supplementary Table S2 and Phusion HighFidelity DNA polymerase (New England Biolabs) and joined together by sequential cloning into vector pUC19 (Vieira & Messing, 1982), giving a deletion construct that still retained the Tat signal peptide and part of the C-terminal peptide coding regions (approximately the last 10 codons of each protein); these constructs were then transferred into the kanamycin-resistant suicide vector pK18mobsacB (Roschanski et al, 2011;Schäfer et al, 1994) and transformed into E. coli S17-1. Kanamycin resistance cassette insertion constructs were created for each B. bacteriovorus tatA1, tatA2 and tatC gene.…”