Lysozymes are among the best-characterized enzymes, acting upon the cell wall substrate peptidoglycan. Here, examining the invasive bacterial periplasmic predator Bdellovibrio bacteriovorus, we ...report a diversified lysozyme, DslA, which acts, unusually, upon (GlcNAc-) deacetylated peptidoglycan. B. bacteriovorus are known to deacetylate the peptidoglycan of the prey bacterium, generating an important chemical difference between prey and self walls and implying usage of a putative deacetyl-specific "exit enzyme". DslA performs this role, and ΔDslA strains exhibit a delay in leaving from prey. The structure of DslA reveals a modified lysozyme superfamily fold, with several adaptations. Biochemical assays confirm DslA specificity for deacetylated cell wall, and usage of two glutamate residues for catalysis. Exogenous DslA, added ex vivo, is able to prematurely liberate B. bacteriovorus from prey, part-way through the predatory lifecycle. We define a mechanism for specificity that invokes steric selection, and use the resultant motif to identify wider DslA homologues.
The bacterium Bdellovibrio bacteriovorus is a predator of other Gram-negative bacteria. The predator invades the prey's periplasm and modifies the prey's cell wall, forming a rounded killed prey, or ...bdelloplast, containing a live B. bacteriovorus. Redundancy in adhesive processes makes invasive mutants rare. Here, we identify a MIDAS adhesin family protein, Bd0875, that is expressed at the predator-prey invasive junction and is important for successful invasion of prey. A mutant strain lacking bd0875 is still able to form round, dead bdelloplasts; however, 10% of the bdelloplasts do not contain B. bacteriovorus, indicative of an invasion defect. Bd0875 activity requires the conserved MIDAS motif, which is linked to catch-and-release activity of MIDAS proteins in other organisms. A proteomic analysis shows that the uninvaded bdelloplasts contain B. bacteriovorus proteins, which are likely secreted into the prey by the Δbd0875 predator during an abortive invasion period. Thus, secretion of proteins into the prey seems to be sufficient for prey killing, even in the absence of a live predator inside the prey periplasm.
Bacterial second messengers are important for regulating diverse bacterial lifestyles. Cyclic di-GMP (c-di-GMP) is produced by diguanylate cyclase enzymes, named GGDEF proteins, which are widespread ...across bacteria. Recently, hybrid promiscuous (Hypr) GGDEF proteins have been described in some bacteria, which produce both c-di-GMP and a more recently identified bacterial second messenger, 3',3'-cyclic-GMP-AMP (cGAMP). One of these proteins was found in the predatory Bdellovibrio bacteriovorus, Bd0367. The bd0367 GGDEF gene deletion strain was found to enter prey cells, but was incapable of leaving exhausted prey remnants via gliding motility on a solid surface once predator cell division was complete. However, it was unclear which signal regulated this process. We show that cGAMP signalling is active within B. bacteriovorus and that, in addition to producing c-di-GMP and some c-di-AMP, Bd0367 is a primary producer of cGAMP in vivo. Site-directed mutagenesis of serine 214 to an aspartate rendered Bd0367 into primarily a c-di-GMP synthase. B. bacteriovorus strain bd0367S214D phenocopies the bd0367 deletion strain by being unable to glide on a solid surface, leading to an inability of new progeny to exit from prey cells post-replication. Thus, this process is regulated by cGAMP. Deletion of bd0367 was also found to be incompatible with wild-type flagellar biogenesis, as a result of an acquired mutation in flagellin chaperone gene homologue fliS, implicating c-di-GMP in regulation of swimming motility. Thus the single Bd0367 enzyme produces two secondary messengers by action of the same GGDEF domain, the first reported example of a synthase that regulates multiple second messengers in vivo. Unlike roles of these signalling molecules in other bacteria, these signal to two separate motility systems, gliding and flagellar, which are essential for completion of the bacterial predation cycle and prey exit by B. bacteriovorus.
Bacterial usage of the cyclic dinucleotide c‐di‐GMP is widespread, governing the transition between motile/sessile and unicellular/multicellular behaviors. There is limited information on c‐di‐GMP ...metabolism, particularly on regulatory mechanisms governing control of EAL c‐di‐GMP phosphodiesterases. Herein, we provide high‐resolution structures for an EAL enzyme Bd1971, from the predatory bacterium Bdellovibrio bacteriovorus, which is controlled by a second signaling nucleotide, cAMP. The full‐length cAMP‐bound form reveals the sensory N‐terminus to be a domain‐swapped variant of the cNMP/CRP family, which in the cAMP‐activated state holds the C‐terminal EAL enzyme in a phosphodiesterase‐active conformation. Using a truncation mutant, we trap both a half‐occupied and inactive apo‐form of the protein, demonstrating a series of conformational changes that alter juxtaposition of the sensory domains. We show that Bd1971 interacts with several GGDEF proteins (c‐di‐GMP producers), but mutants of Bd1971 do not share the discrete phenotypes of GGDEF mutants, instead having an elevated level of c‐di‐GMP, suggesting that the role of Bd1971 is to moderate these levels, allowing “action potentials” to be generated by each GGDEF protein to effect their specific functions.
Synopsis
The cyclic‐di‐GMP hydrolase Bd1971 is a sensor‐enzyme fusion protein controlling turnover and predatory behavior of the bacterium Bdellovibrio bacteriovorus. Here, structural work combined with enzyme assays and genetics reveal regulation of Bd1971 by cAMP, with important consequences for signalling during the Bdellovibrio lifecycle.
The Bd1971 structure identifies a sensor adapted from the dimeric CRP superfamily.
Bd1971 bound to stimulus cAMP renders the protein active to hydrolyse cyclic‐di‐GMP.
Empty and part‐occupied sensor structures reveal a “swing‐out” mechanism of regulation.
Bd1971 can bind GGDEF synthases and putatively allow “action potential” spikes of cyclic‐di‐GMP to be generated.
Structural and biochemical work shows how usage of the second messenger cyclic dinucleotide c‐di‐GMP is controlled by upstream cAMP.
The predatory bacterium
grows and divides inside the periplasm of Gram-negative bacteria, forming a structure known as a bdelloplast. Cell division of predators inside the dead prey cell is not by ...binary fission but instead by synchronous division of a single elongated filamentous cell into odd or even numbers of progeny cells.
replication and cell division processes are dependent on the finite level of nutrients available from inside the prey bacterium. The filamentous growth and division process of the predator maximizes the number of progeny produced by the finite nutrients in a way that binary fission could not. To learn more about such an unusual growth profile, we studied the role of DivIVA in the growing
cell. This protein is well known for its link to polar cell growth and spore formation in Gram-positive bacteria, but little is known about its function in a predatory growth context. We show that DivIVA is expressed in the growing
cell and controls cell morphology during filamentous cell division, but not the number of progeny produced. Bacterial Two Hybrid (BTH) analysis shows DivIVA may interact with proteins that respond to metabolic indicators of amino-acid biosynthesis or changes in redox state. Such changes may be relevant signals to the predator, indicating the consumption of prey nutrients within the sealed bdelloplast environment. ParA, a chromosome segregation protein, also contributes to bacterial septation in many species. The
genome contains three ParA homologs; we identify a canonical ParAB pair required for predatory cell division and show a BTH interaction between a gene product encoded from the same operon as DivIVA with the canonical ParA. The remaining ParA proteins are both expressed in
but are not required for predator cell division. Instead, one of these ParA proteins coordinates gliding motility, changing the frequency at which the cells reverse direction. Our work will prime further studies into how one bacterium can co-ordinate its cell division with the destruction of another bacterium that it dwells within.
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 MglA(Bd) GTP-binding are conserved. Deletion of mglA(Bd) abolished prey-invasion, but not gliding, and reduced T4P formation. MglA(Bd) 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, RomR(Bd) 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.
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 prey-independent 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 backgrounds.
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 ~1x10(7) 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.
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.
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.
Summary
The predatory bacterium Bdellovibrio bacteriovorus swims rapidly by rotation of a single, polar flagellum comprised of a helical filament of flagellin monomers, contained within a membrane ...sheath and powered by a basal motor complex. Bdellovibrio collides with, enters and replicates within bacterial prey, a process previously suggested to firstly require flagellar motility and then flagellar shedding upon prey entry. Here we show that flagella are not always shed upon prey entry and we study the six fliC flagellin genes of B. bacteriovorus, finding them all conserved and expressed in genome strain HD100 and the widely studied lab strain 109J. Individual inactivation of five of the fliC genes gave mutant Bdellovibrio that still made flagella, and which were motile and predatory. Inactivation of the sixth fliC gene abolished normal flagellar synthesis and motility, but a disordered flagellar sheath was still seen. We find that this non‐motile mutant was still able to predate when directly applied to lawns of YFP‐labelled prey bacteria, showing that flagellar motility is not essential for prey entry but important for efficient encounters with prey in liquid environments.
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