The first bite--profiling the predatosome in the bacterial pathogen Bdellovibrio

Abstract

Bdellovibrio bacteriovorus is a Gram-negative bacterium that is a pathogen of other Gram-negative bacteria, including many bacteria which are pathogens of humans, animals and plants. As such Bdellovibrio has potential as a biocontrol agent, or living antibiotic. B. bacteriovorus HD100 has a large genome and it is not yet known which of it encodes the molecular machinery and genetic control of predatory processes. We have tried to fill this knowledge-gap using mixtures of predator and prey mRNAs to monitor changes in Bdellovibrio gene expression at a timepoint of early-stage prey infection and prey killing in comparison to control cultures of predator and prey alone and also in comparison to Bdellovibrio growing axenically (in a prey-or host independent "HI" manner) on artificial media containing peptone and tryptone. From this we have highlighted genes of the early predatosome with predicted roles in prey killing and digestion and have gained insights into possible regulatory mechanisms as Bdellovibrio enter and establish within the prey bdelloplast. Approximately seven percent of all Bdellovibrio genes were significantly up-regulated at 30 minutes of infection--but not in HI growth--implicating the role of these genes in prey digestion. Five percent were down-regulated significantly, implicating their role in free-swimming, attack-phase physiology. This study gives the first post-genomic insight into the predatory process and reveals some of the important genes that Bdellovibrio expresses inside the prey bacterium during the initial attack.

Figure 1. Predatory and HI growth cycles of Bdellovibrio.

Attack-phase cells invading E. coli prey undergo a predatory growth cycle and replicate, being released some 4 hours later. Timing of the predatory cycle events are shown, including the 30 minute predation stage when Bdellovibrio are in the bdelloplast. Axenic growth of pleiomorphic sessile HI cells occurs on protein-rich media for cells derived from attack-phase populations.

Figure 2. Combining Attack-phase, HI and Predatory Datasets.

Venn diagram produced by combining our datasets in a comparison of genes up or down-regulated in 30 minutes predation versus attack-phase and those up or down regulated in HI growth versus attack phase. This allows identification of gene expression patterns specific to each growth state- 30 minutes predation, attack-phase outside prey, or axenic HI growth without prey. For example 240 genes that are specifically up-regulated only upon 30 minutes predation, compared to attack phase; are delineated away from a set, of 239 “release from starvation and growth initiation” genes that are up-regulated both in predation and HI growth, compared to non-replicative attack-phase Bdellovibrio.

Figure 3. Up- and Down-regulated genes from attack-phase to predatory come from very different KEGG categories.

Pie charts showing the very different category distributions, of significantly down regulated or up-regulated Bdellovibrio genes upon the shift from attack phase to 30 minutes predation. (Only genes that have KEGG categories are shown, organised by those categories). Type iii secretion system category refers only to flagellar genes as Bdellovibrio has no other Type III system).

Figure 4. RT-PCR to show that differential expression is not HI-strain specific.

RT-PCR on matched amounts of RNA to compare host-dependent attack phase (sample 1) and 4 hours post-infection (2) with 4 different HI strains HID13 (3), HID2 (4), HID22 (5) and HID26 (6) along with controls of E. coli prey RNA (7), no template (8) and HD100 genomic DNA as template (9). Primers were designed against 2 genes upregulated in HI phase (narL and Bd1640), 2 genes constitutively expressed in both phases (dnaK and Bd0881) and 2 genes with lower expression in the HI phase (Bd2462 and Bd2439). The fact that all 4 strains show the same expression patterns confirms that the results obtained were a consequence of the HI phenotype in general and not specific to the strain HID13 used for the arrays.

Figure 5. QRT-PCR results confirming the array results displayed on a log scale.

T-test on the QRT-PCR data also showed that sdhB and pilA were not significantly differentially regulated in the transition from attack phase to predatory conditions and that dnaK and Bd1168 were not significantly differentially regulated in the transition from attack phase to HI conditions. It shows that Bd1904, Bd0416, Bd0659, and Bd2620 were significantly differentially regulated in the transition from attack phase to predatory and that narL, Bd1476, Bd2462 and Bd0367 were significantly differentially regulated in the transition from attack phase to HI. (T-test results confirmed the differences in expression between differentially regulated genes to be significant and that there were no significant differences in expression levels for those genes called as not differentially expressed in the array data). Darker colours are for genes confirming the HI array data, lighter colours for the 30 minutes predation experiment.

Figure 6. Transcription profiles of upregulated hypothetical genes across the predatory cycle.

RT-PCR on total RNA prepared from identical volumes of B. bacteriovorus HD100 predator-prey E. coli S17-1 infection culture as the predatory infection proceeds across a time course. Primers were designed against three Bdellovibrio-specific but otherwise unknown function that specify proteins with signal sequences. These genes were shown by the array studies to be significantly up-regulated upon the shift from attack-phase to 30 minutes of predatory growth. Lanes 1 & 14 NEB 100bp ladder, 2: attack-phase 3: 15 minutes predation, 4: 30 minutes predation, 5: 45 minutes predation 6–9: 1,2,3,4 hours predation respectively 10: HI growth, 11: no template control 12: E. coli S17-1 only RNA control 13 B. bacteriovorus HD100 genomic DNA control.

Figure 7. Gene expression key to attack phase.

Visual summaries of key gene expression characteristics of the attack phase of Bdellovibrio cells, as implied by genes highly expressed In the attack phase dataset, but downregulated in the other two datasets.

Figure 8. Gene expression key to 30 minutes predation.

Visual summaries of key gene expression characteristics at 30 minutes of predatory growth by Bdellovibrio cells, as implied by genes highly expressed in the predatory dataset, but downregulated in the other two datasets.

Figure 9. Gene expression key to HI growth phase.

Visual summaries of key gene expression characteristics of the HI growth phase of Bdellovibrio cells, as implied by genes highly expressed in the HI dataset, but downregulated in the other two datasets.