Genome analysis of a simultaneously predatory and prey-independent, novel Bdellovibrio bacteriovorus from the River Tiber, supports in silico predictions of both ancient and recent lateral gene transfer from diverse bacteria

Background: Evolution equipped Bdellovibrio bacteriovorus predatory bacteria to invade other bacteria, digesting and replicating, sealed within them thus preventing nutrient-sharing with organisms in the surrounding environment. Bdellovibrio were previously described as “obligate predators” because only by mutations, often in gene bd0108, are 1 in ~1x107 of predatory lab strains of Bdellovibrio converted to prey-independent growth. A previous genomic analysis of B. bacteriovorus strain HD100 suggested that predatory consumption of prey DNA by lytic enzymes made Bdellovibrio less likely than other bacteria to acquire DNA by lateral gene transfer (LGT). However the Doolittle and Pan groups predicted, in silico, both ancient and recent lateral gene transfer into the B. bacteriovorus HD100 genome. Results: To test these predictions, we isolated a predatory bacterium from the River Tiber- a good potential source of LGT as it is rich in diverse bacteria and organic pollutants- by enrichment culturing with E. coli prey cells. The isolate was identified as B. bacteriovorus and named as strain Tiberius. Unusually, this Tiberius strain showed simultaneous prey-independent growth on organic nutrients and predatory growth on live prey. Despite the prey-independent growth, the homolog of bd0108 did not have typical prey-independent-type mutations. The dual growth mode may reflect the high carbon content of the river, and gives B. bacteriovorus Tiberius extended non-predatory contact with the other bacteria present. The HD100 and Tiberius genomes were extensively syntenic despite their different cultured-terrestrial/freshly-isolated aquatic histories; but there were significant differences in gene content indicative of genomic flux and LGT. Gene content comparisons support previously published in silico predictions for LGT in strain HD100 with substantial conservation of genes predicted to have ancient LGT origins but little conservation of AT-rich genes predicted to be recently acquired. Conclusions: The natural niche and dual predatory, and prey-independent growth of the B. bacteriovorus Tiberius strain afforded it extensive non-predatory contact with other marine and freshwater bacteria from which LGT is evident in its genome. Thus despite their arsenal of DNA-lytic enzymes; Bdellovibrio are not always predatory in natural niches and their genomes are shaped by acquiring whole genes from other bacteria

Recommendations for a core outcome set for measuring standing balance in adult populations: a consensus-based approach

Standing balance is imperative for mobility and avoiding falls. Use of an excessive number of standing balance measures has limited the synthesis of balance intervention data and hampered consistent clinical practice.To develop recommendations for a core outcome set (COS) of standing balance measures for research and practice among adults.A combination of scoping reviews, literature appraisal, anonymous voting and face-to-face meetings with fourteen invited experts from a range of disciplines with international recognition in balance measurement and falls prevention. Consensus was sought over three rounds using pre-established criteria.The scoping review identified 56 existing standing balance measures validated in adult populations with evidence of use in the past five years, and these were considered for inclusion in the COS.Fifteen measures were excluded after the first round of scoring and a further 36 after round two. Five measures were considered in round three. Two measures reached consensus for recommendation, and the expert panel recommended that at a minimum, either the Berg Balance Scale or Mini Balance Evaluation Systems Test be used when measuring standing balance in adult populations.Inclusion of two measures in the COS may increase the feasibility of potential uptake, but poses challenges for data synthesis. Adoption of the standing balance COS does not constitute a comprehensive balance assessment for any population, and users should include additional validated measures as appropriate.The absence of a gold standard for measuring standing balance has contributed to the proliferation of outcome measures. These recommendations represent an important first step towards greater standardization in the assessment and measurement of this critical skill and will inform clinical research and practice internationally

Ras GTPase-like protein MglA, a controller of bacterial social-motility in Myxobacteria, has evolved to control bacterial predation by Bdellovibrio

Bdellovibrio bacteriovorus invade Gram-negative bacteria in a predatory process requiring Type IV pili (T4P) at a single invasive pole, and also glide on surfaces to locate prey. Ras-like G-protein MglA, working with MglB and RomR in the deltaproteobacterium Myxococcus xanthus, regulates adventurous gliding and T4P-mediated social motility at both M. xanthus cell poles. Our bioinformatic analyses suggested that the GTPase activating protein (GAP)-encoding gene mglB was lost in Bdellovibrio, but critical residues for MglABd GTP-binding are conserved. Deletion of mglABd abolished prey-invasion, but not gliding, and reduced T4P formation. MglABd interacted with a previously uncharacterised tetratricopeptide repeat (TPR) domain protein Bd2492, which we show localises at the single invasive pole and is required for predation. Bd2492 and RomR also interacted with cyclic-di-GMP-binding receptor CdgA, required for rapid prey-invasion. Bd2492, RomRBd and CdgA localize to the invasive pole and may facilitate MglA-docking. Bd2492 was encoded from an operon encoding a TamAB-like secretion system. The TamA protein and RomR were found, by gene deletion tests, to be essential for viability in both predatory and non-predatory modes. Control proteins, which regulate bipolar T4P-mediated social motility in swarming groups of deltaproteobacteria, have adapted in evolution to regulate the anti-social process of unipolar prey-invasion in the “lone-hunter” Bdellovibrio. Thus GTP-binding proteins and cyclic-di-GMP inputs combine at a regulatory hub, turning on prey-invasion and allowing invasion and killing of bacterial pathogens and consequent predatory growth of Bdellovibrio

Discrete cyclic di-GMP-dependent control of bacterial predation versus axenic growth in Bdellovibrio bacteriovorus

Bdellovibrio bacteriovorus is a Delta-proteobacterium that oscillates between free-living growth and predation on Gram-negative bacteria including important pathogens of man, animals and plants. After entering the prey periplasm, killing the prey and replicating inside the prey bdelloplast, several motile B. bacteriovorus progeny cells emerge. The B. bacteriovorus HD100 genome encodes numerous proteins predicted to be involved in signalling via the secondary messenger cyclic di-GMP (c-di-GMP), which is known to affect bacterial lifestyle choices. We investigated the role of c-di-GMP signalling in B. bacteriovorus, focussing on the five GGDEF domain proteins that are predicted to function as diguanylyl cyclases initiating c-di-GMP signalling cascades. Inactivation of individual GGDEF domain genes resulted in remarkably distinct phenotypes. Deletion of dgcB (Bd0742) resulted in a predation impaired, obligately axenic mutant, while deletion of dgcC (Bd1434) resulted in the opposite, obligately predatory mutant. Deletion of dgcA (Bd0367) abolished gliding motility, producing bacteria capable of predatory invasion but unable to leave the exhausted prey. Complementation was achieved with wild type dgc genes, but not with GGAAF versions. Deletion of cdgA (Bd3125) substantially slowed predation; this was restored by wild type complementation. Deletion of dgcD (Bd3766) had no observable phenotype. In vitro assays showed that DgcA, DgcB, and DgcC were diguanylyl cyclases. CdgA lacks enzymatic activity but functions as a c-di-GMP receptor apparently in the DgcB pathway. Activity of DgcD was not detected. Deletion of DgcA strongly decreased the extractable c-di-GMP content of axenic Bdellovibrio cells. We show that c-di-GMP signalling pathways are essential for both the free-living and predatory lifestyles of B. bacteriovorus and that obligately predatory dgcC- can be made lacking a propensity to survive without predation of bacterial pathogens and thus possibly useful in anti-pathogen applications. In contrast to many studies in other bacteria, Bdellovibrio shows specificity and lack of overlap in c-di-GMP signalling pathways

Model for <i>B. bacteriovorus</i> predatory-pole regulation during prey-invasion and its relationship to <i>M. xanthus</i> bipolar motility-control proteins.

<p>During prey-invasion; TamA_Bd, RomR_Bd and CdgA protein interactions occur (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004253#pgen.1004253.s002" target="_blank">Figure S2</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004253#pgen.1004253.s003" target="_blank">S3 </a><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004253#pgen.1004253.s004" target="_blank">S4</a>) at the single <i>B. bacteriovorus</i> pole. This could control localization of the TamA_Bd-like OMP at the prey-interaction pole or activate it to receive, (via its POTRA domains), and secrete predatory outer membrane or autotransporter proteins. The action of this secretion via Bd2495 TamA is essential to both predatory and HI lifestyles, and RomR_Bd, (which is also essential), may regulate or report the activity of the TamAB transport system, at the single predatory pole. Additional regulation of this activity could be influenced by c-di-GMP for which CdgA, (another hub protein that binds RomR and TPR Bd2492), is a receptor in <i>Bdellovibrio</i>. MglA_Bd interacts with Bd2492 at the predatory pole but also is found more diffusely in the cell. MglA interactions may regulate prey entry via TPR Bd2492, as deletion of MglA or TPR Bd2492 abolishes prey-invasion but not prey attachment. MglA deletion in <i>Bdellovibrio</i> greatly reduces the level of Type IV pilus formation at the single anterior pole. In contrast Keilberg and co-workers showed that <i>M. xanthus</i> RomR and MglB localise bipolarly asymmetrically, while MglA typically localises at the leading cell pole, during surface movement, to regulate both A- and S- motility (<i>M. xanthus</i> after <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004253#pgen.1004253-Keilberg1" target="_blank">[23]</a>).</p

<i>B. bacteriovorus</i> RomR-mCherry and Bd2492-mCherry localised at the prey-interaction pole; MglA-mCherry showed variable diffuse foci.

<p><i>B. bacteriovorus</i> cells were incubated with E. coli S17-1 prey cells for 5 minutes, allowing sufficient time for some of the <i>Bdellovibrio</i> cells to attach to prey. Panels- <b>A</b>: The lower prey-cell shows a typical attached <i>Bdellovibrio</i> cell, with a RomR-mCherry focus at the anterior (attached) pole of the <i>Bdellovibrio</i>. <b>B</b>: The rightmost prey-cell shows a typical attached <i>Bdellovibrio</i> cell, with a Bd2492-mCherry focus at the anterior (attached) pole of the <i>Bdellovibrio</i>. <b>C</b>: MglA-mCherry <i>Bdellovibrio</i> cells had variable foci, including diffuse and unipolar localisations. From left to right, all panels show brightfield, fluorescent, and merged images and a graphical representation. Fluorescent exposure = 2 seconds.</p

Host-independent invasion and attachment assays of Δ<i>mglA</i> strain and wild-type controls.

<p>(A) <i>Attachment assay</i>: After 1 hour, 21.0% of <i>E. coli</i> cells were attached to and invaded by wild-type <i>Bdellovibrio</i> HI strain HID26 cells. A further 22.5% of <i>E. coli</i> cells were attached to, but not invaded by HID26 cells. 23.0% and 17.4% of <i>E. coli</i> cells were attached to by Δ<i>mglA</i> HI and Δ<i>pilA</i> HI, respectively. The Δ<i>mglA</i> HI and Δ<i>pilA</i> HI strains never invaded to form bdelloplasts. The attachment assay has the following variability: Percentage points (pp): WT 43.5%±15.5 pp; Δ<i>mglA</i> 23.0%±4.2 pp; Δ<i>pilA</i> 17.4%±7.3 pp. (B) <i>Invasion assay</i>: HI wild-type <i>Bdellovibrio</i> control HID50 was able to infect <i>E. coli</i> prey cells (26.3% of <i>E. coli</i> cells invaded to form bdelloplasts after 22 hours, 45/171 <i>E. coli</i> cells). <i>Bdellovibrio</i> Δ<i>mglA</i> HI strain could not invade <i>E. coli</i> prey (0.0% of <i>E. coli</i> cells invaded to form bdelloplasts after 22 hours, 0/319 <i>E. coli</i> cells). Fluorescent images show representative fluorescent <i>E.coli</i> S17-1::pMAL_p2-mCherry cells, either uninfected or rounded to form bdelloplasts.</p

Gene synteny of <i>bd2492–bd2495</i> homologues is conserved in <i>B. bacteriovorus</i>, <i>M. xanthus</i> and <i>B. marinus</i>.

<p>Genes encoding a TPR domain protein are followed by genes encoding a DUF490 domain protein and an Omp85 superfamily protein in all three bacterial species. In <i>M. xanthus</i>, the three genes are interrupted by a gene encoding a putative Sec system ATPase, MXAN_5765. Percentage protein sequence identities and similarities with the <i>B. bacteriovorus</i> protein (NEEDLE global alignment) are shown underneath (for operon-confirmation of <i>bd2492-2495</i> see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004253#pgen.1004253.s006" target="_blank">Figure S6</a>).</p

Predation and <i>in cis</i> complementation of <i>B. bacteriovorus <i>Δ</i>mglA</i> HI strains, on <i>E. coli</i> prey.

<p>(A) Predation efficiency of the Δ<i>mglA</i> HI strain was assayed against predatory and non-predatory controls by the reduction of <i>E. coli</i> numbers over 48 hours. Three wild-type HI strains (HID13, HID26 and HID50) reduced <i>E. coli</i> numbers in liquid cultures by up to four logs (grey region shows known natural variation in predation rate between different wild-type HI isolates). The Δ<i>mglA</i> HI strain showed no reduction in <i>E. coli</i> numbers, comparable to a previously-studied, non-predatory Δ<i>pilA</i> HI strain, and to <i>E. coli</i> with no added <i>B. bacteriovorus</i>. (B) Reintroduction of the <i>mglA</i> ORF <i>in cis</i> to the Δ<i>mglA</i> HI strain in plasmid pK18::<i>mglA</i> restored predatory growth. Error bars represent 1 SD from the mean (for predation-testing of <b>Δ</b><i>bd2492</i> strain see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004253#pgen.1004253.s005" target="_blank">Figure S5</a>).</p

Protein alignment of MglA_Mx and MglA_Bd.

<p>MglA_Mx (MXAN1925) alignment with MglA_Bd (Bd3734) shows significant sequence similarity between the two proteins. (A) The P-loop is conserved in <i>B. bacteriovorus</i>, although a serine is present in place of a glycine residue at position 21 (signified by arrow). The PM1/G1 threonine residue (B) and PM3 (C); and G2 (D) motifs are all conserved between the two proteins (for <i>mglA</i> genes, encoding MglA G21, co-occurring with <i>mglB</i> genes see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004253#pgen.1004253.s001" target="_blank">Figure S1</a>).</p