A Transcriptional “Scream” Early Response of E. coli Prey to Predatory Invasion by Bdellovibrio

We have transcriptionally profiled the genes differentially expressed in E. coli prey cells when predatorily attacked by Bdellovibrio bacteriovorus just prior to prey cell killing. This is a brief, approximately 20–25 min period when the prey cell is still alive but contains a Bdellovibrio cell in its periplasm or attached to and penetrating its outer membrane. Total RNA was harvested and labelled 15 min after initiating a semi-synchronous infection with an excess of Bdellovibrio preying upon E. coli and hybridised to a macroarray spotted with all predicted ORFs of E. coli. SAM analysis and t-tests were performed on the resulting data and 126 E. coli genes were found to be significantly differentially regulated by the prey upon attack by Bdellovibrio. The results were confirmed by QRT-PCR. Amongst the prey genes upregulated were a variety of general stress response genes, potentially “selfish” genes within or near prophages and transposable elements, and genes responding to damage in the periplasm and osmotic stress. Essentially, the presence of the invading Bdellovibrio and the resulting damage to the prey cell elicited a small “transcriptional scream”, but seemingly no specific defensive mechanism with which to counter the Bdellovibrio attack. This supports other studies which do not find Bdellovibrio resistance responses in prey, and bodes well for its use as a “living antibiotic”

Activity of Bdellovibrio Hit Locus Proteins, Bd0108 and Bd0109, Links Type IVa Pilus Extrusion/Retraction Status to Prey-Independent Growth Signalling

Bdellovibrio bacteriovorus are facultatively predatory bacteria that grow within gram-negative prey, using pili to invade their periplasmic niche. They also grow prey-independently on organic nutrients after undergoing a reversible switch. The nature of the growth switching mechanism has been elusive, but several independent reports suggested mutations in the hit (host-interaction) locus on the Bdellovibrio genome were associated with the transition to preyindependent growth. Pili are essential for prey entry by Bdellovibrio and sequence analysis of the hit locus predicted that it was part of a cluster of Type IVb pilus-associated genes, containing bd0108 and bd0109. In this study we have deleted the whole bd0108 gene, which is unique to Bdellovibrio, and compared its phenotype to strains containing spontaneous mutations in bd0108 and the common natural 42 bp deletion variant of bd0108. We find that deletion of the whole bd0108 gene greatly reduced the extrusion of pili, whereas the 42 bp deletion caused greater pilus extrusion than wild-type. The pili isolated from these strains were comprised of the Type IVa pilin protein; PilA. Attempts to similarly delete gene bd0109, which like bd0108 encodes a periplasmic/secreted protein, were not successful, suggesting that it is likely to be essential for Bdellovibrio viability in any growth mode. Bd0109 has a sugar binding YD- repeat motif and an N-terminus with a putative pilin-like fold and was found to interact directly with Bd0108. These results lead us to propose that the Bd0109/Bd0108 interaction regulates pilus production in Bdellovibrio (possibly by interaction with the pilus fibre at the cell wall), and that the presence (and possibly retraction state) of the pilus feeds back to alter the growth state of the Bdellovibrio cell. We further identify a novel small RNA encoded by the hit locus, the transcription of which is altered in different bd0108 mutation background

Manipulating Each MreB of Bdellovibrio bacteriovorus Gives Diverse Morphological and Predatory Phenotypes▿

We studied the two mreB genes, encoding actinlike cytoskeletal elements, in the predatory bacterium Bdellovibrio bacteriovorus. This bacterium enters and replicates within other Gram-negative bacteria by attack-phase Bdellovibrio squeezing through prey outer membrane, residing and growing filamentously in the prey periplasm forming an infective “bdelloplast,” and septating after 4 h, once the prey contents are consumed. This lifestyle brings challenges to the Bdellovibrio cytoskeleton. Both mreB genes were essential for viable predatory growth, but C-terminal green fluorescent protein tagging each separately with monomeric teal-fluorescent protein (mTFP) gave two strains with phenotypic changes at different stages in predatory growth and development. MreB1-mTFP cells arrested growth early in bdelloplast formation, despite successful degradation of prey nucleoid. A large population of stalled bdelloplasts formed in predatory cultures and predation proceeded very slowly. A small proportion of bdelloplasts lysed after several days, liberating MreB1-mTFP attack-phase cells of wild-type morphology; this process was aided by subinhibitory concentrations of an MreB-specific inhibitor, A22. MreB2-mTFP, in contrast, was predatory at an almost wild-type rate but yielded attack-phase cells with diverse morphologies, including spherical, elongated, and branched, the first time such phenotypes have been described. Wild-type predatory rates were seen for all but spherical morphotypes, and septation of elongated morphotypes was achieved by the addition of A22

Predation of HI strains compared to predatory HD100.

<div><p>Time-lapse microscopy of individual wild-type HD100 cells (<b>A</b>(<b>a</b>)) and cells of the markerless deletion mutant of <i>bd0108</i> (A(b)) preying upon the <i>E. coli</i> strain pMAL-p2_mCherry with a fluorescent periplasm. At T=0 there is attachment of free swimming <i>Bdellovibrio</i> in both strains, with invasion occurring within 2 frames (5 minutes) for most cells observed. Arrows indicate both free swimming and attached <i>Bdellovibrio</i> cells. By 1 hour the <i>Bdellovibrio</i> is established in the periplasm and begun to grow as a filament. By 8 hours the growing filaments of both the wild-type and deletion strains have septated to form single progeny and the prey bursts releasing them. Predation was carried out on a 1% Agarose pad with images acquired every 2.5 minutes.</p> <p><b>B</b>. Reduction of <i>E. coli</i> numbers in a predatory lysate comparing wild-type HD100 or spontaneously generated HIs with the markerless deletion of <i>bd0108</i> strains. The spontaneously generated HI strains included some with a variety of point mutations or the common 42bp deletion in <i>bd0108</i> and some with a wild-type copy of <i>bd0108</i>. HI cultures readily prey upon <i>E. coli</i> in liquid cultures, reducing prey numbers by several logs in 2 days with predation by the population as a whole appearing to be slightly slower than wild-type HD100. All HI strains were grown axenically, independently of prey cells before the experiment. </p></div

Luminescent prey assay of predation efficiency for Host Dependent and Host Independent strains.

<p>For Host Dependent cells (<b>A</b>) with a genomic copy of the wild-type <i>bd0108</i> gene, carrying the pSUP404.2 plasmid encoding either the wild-type <i>bd0108</i> gene or the 42 bp deletion variant of <i>bd0108</i> had no effect on predation compared to those carrying the empty pSUP404.2 plasmid. This assay shows the typical result of logarithmically faster reduction in luminescence with more <i>Bdellovibrio</i> initially added and shows that the extra copies of <i>bd0108</i> or the 42 bp deletion variant have no significant effect on this. For Host Independent cells (<b>B</b>) there is not the same proportional decrease in luminescence when more cells are added supporting the hypothesis that a proportion are growing axenically as HIs rather than predatorily. HI mutants carrying the pSUP404.2 plasmid encoding the wild-type <i>bd0108</i> gene, however, restore the typical initial-cell-number to drop in luminescence relationship seen in the HD wild-type. This suggests that the presence of a wild-type <i>bd0108</i>, but not the 42 bp deletion variant, <i>in </i><i>trans</i> restores the HI cells to a predatory lifestyle.</p

RT-PCR expression of the sRNA downstream of <i>bd0108</i> and expression of a control gene <i>dnaK</i>.

<p>Expression of a small, non-coding RNA downstream of <i>bd0108</i> was shown to be different in HI strains carrying different mutations in <i>bd0108</i> and attack phase HD100. Expression was highest in HID13 (ATA->ATG start codon mutation) and the strains with the markerless deletion of <i>bd0108</i>. Strain HID22 (<i>bd0108∆</i>42bp), had slightly lower expression, while those strains with a wild-type <i>bd0108</i>; HD100 and HID2, show much lower expression of the sRNA. Expression of <i>dnaK</i> was uniform across the samples indicating a matched amount of total RNA was used for the experiment.</p

PFU to CFU ratio for pooled spontaneous HI strains and the markerless <i>bd0108</i> deletion HI mutants of <i>Bdellovibrio</i>.

<p>In the spontaneous generated HI strains; HID2 and HID26 (wild-type for <i>bd0108</i>) HID6, HID13, HID18, (point mutations in <i>bd0108</i>) and HID22 (<i>bd0108∆</i>42bp), and the markerless deletion mutants of <i>bd0108</i>, complementation tests shows that these strains carrying the pSUP404.2 plasmid encoding the wild-type <i>bd0108</i> is significantly deficient in the capacity to form HI colonies on PY agar plates compared to the capacity to form plaques on double layer overlay plates. This was significant compared to cells containing the pSUP404.2 plasmid alone, or the pSUP404.2-Δ42 with the 42 bp deletion variant of <i>bd0108</i>. The ratio of CFU:PFU was reduced from 2.16 x 10^-1 in strains containing the pSUP404.2 alone to 3.21 x 10^-2 in strains containing pSUP404.2-108 (p = 0.026) but were not significantly reduced relative to strains containing pSUP404.2-Δ42, 2.56 x 10^-1 (p = 0.31). The difference between strains carrying the pSUP404.2-108 and the pSUP404.2-Δ42 was also significant (P<<0.01).</p

Model for possible interactions of Bd0108/Bd0109 controlling the extrusion and retraction of pili.

<div><p>A. Operonal structure of the <i>bd0108 </i><i>hit</i> locus and surrounding genes, predicted to have a role in the formation of a Type IVb pilus. Genes are colour coded to correspond to their predicted function in the pilus diagrams underneath.</p> <p><b>B</b>. In wild-type cells <i>bd0108</i> and <i>bd0109</i> are co-expressed, the mRNA is then translated into proteins containing a signal sequence recognised by the Sec system, the signal is cleaved, and the proteins are transported into the periplasm where the mature Bd0108 protein transiently interacts with Bd0109 to sequester it. When Bd0109 is unbound, it could then anchor at either the cell wall, or with the mature pilus fibre. Both scenarios are possible due to Bd0109’s structural cleft binding carbohydrate that is present in both cell wall and the mature and glycosylated pili. Bd0109 mediates successful pilus extrusion/retraction and signal back into the cytoplasm. In wild-type pilus formation Bd1290 pre-pilins are held in the inner membrane and are assembled into the pilus fibre possibly by the flp pilus ATPases Bd0110 and Bd0111. The balance of sequestering and release of Bd0109 by Bd0108 in the periplasm permits to successful extrusion and retraction of the pilus fibre upon environmental cues.</p> <p><b>C</b>. In the absence of Bd0108 protein, Bd0109 is not sequestered and is free to mediate more frequently with pilus extrusion and retraction, resulting in very few pili extruded beyond the cell surface and cues for HI growth signalled to the cell.</p> <p><b>D</b>. In HI strains containing the 42 bp deletion variant of <i>bd0108</i>, the gene is still expressed. The truncated form of Bd0108 alters the dynamics of the Bd0109 functionalisation are altered (possibly by over-sequestration of Bd0109) and hyper-extruded pili are seen on the surface more frequently. Hyper-extruded pili or no pili send similar internal signals to regulate prey independent growth.</p></div

<i>In trans</i> expression of fluorescently tagged Bd0108 and Bd0109 in <i>E. coli</i> S17-1 heterologous host.

<div><p><b>A</b>. <i>E. coli</i> S17-1 expressing a C-terminal mCherry fusion of Bd0108 protein <i>in </i><i>trans</i>. Fluorescence can be seen in the periplasm of the cells, with large amounts towards the poles of the cell where cells are plasmolysed.</p> <p><b>B</b>. <i>E. coli</i> S17-1 expressing a C-terminal mTFP fusion of Bd0109 protein <i>in </i><i>trans</i>. Fluorescence can be seen in the periplasm of the cells, with large amounts towards the poles of the cell where cells are plasmolysed.</p></div