Nucleases in Bdellovibrio bacteriovorus contribute towards efficient self-biofilm formation and eradication of preformed prey biofilms

Bdellovibrio bacteriovorus are predatory bacteria that burrow into prey bacteria and degrade their cell contents, including DNA and RNA, to grow. Their genome encodes diverse nucleases, some with potential export sequences. Transcriptomic analysis determined two candidate-predicted nuclease genes (bd1244, bd1934) upregulated upon contact with prey, which we hypothesised, may be involved in prey nucleic acid degradation. RT-PCR on total RNA from across the predatory cycle confirmed that the transcription of these genes peaks shortly after prey cell invasion, around the time that prey DNA is being degraded. We deleted bd1244 and bd1934 both singly and together and investigated their role in predation of prey cells and biofilms. Surprisingly, we found that the nuclease-mutant strains could still prey upon planktonic bacteria as efficiently as wild type and still degraded the prey genomic DNA. The Bdellovibrio nuclease mutants were less efficient at (self-) biofilm formation, and surprisingly, they showed enhanced predatory clearance of preformed prey cell biofilms relative to wild-type Bdellovibrio

Nucleases in Bdellovibrio bacteriovorus contribute towards efficient self-biofilm formation and eradication of preformed prey biofilms

Bdellovibrio bacteriovorus are predatory bacteria that burrow into prey bacteria and degrade their cell contents, including DNA and RNA, to grow. Their genome encodes diverse nucleases, some with potential export sequences. Transcriptomic analysis determined two candidate-predicted nuclease genes (bd1244, bd1934) upregulated upon contact with prey, which we hypothesised, may be involved in prey nucleic acid degradation. RT-PCR on total RNA from across the predatory cycle confirmed that the transcription of these genes peaks shortly after prey cell invasion, around the time that prey DNA is being degraded. We deleted bd1244 and bd1934 both singly and together and investigated their role in predation of prey cells and biofilms. Surprisingly, we found that the nuclease-mutant strains could still prey upon planktonic bacteria as efficiently as wild type and still degraded the prey genomic DNA. The Bdellovibrio nuclease mutants were less efficient at (self-) biofilm formation, and surprisingly, they showed enhanced predatory clearance of preformed prey cell biofilms relative to wild-type Bdellovibrio

Transcriptional analysis of Bdellovibrio bacteriovorus responses to different prey and environments

Bdellovibrio bacteriovorus is a small, highly motile Gram-negative bacterium that preys upon other Gram-negative bacteria. It does this by burrowing through the outer layers of the prey cell and establishing itself in the periplasm, before consuming the prey contents and using these for growth as a filament. When the prey contents are depleted, this filament septates to produce new B. bacteriovorus progeny which burst out of the prey shell and go on to attack further prey. Its prey includes pathogens of plants, animals and humans, including multidrug-resistant pathogens which are emerging as a major health threat. Thus it has potential as a novel therapeutic to overcome the lack of new antibiotics. This prospect is particularly attractive as genetic resistance to B. bacteriovorus predation has not been demonstrated, rather a plastic resistance to a sub-population is seen, which when recovered remains as susceptible to predation as the parent population. There are many stages to this complex predatory lifecycle: swimming or gliding to search out potential prey, attachment to and detecting suitable prey, formation of, and entry into, a pore in the outer layers which is then re-sealed. Then killing and rounding of the prey is followed by staged degradation of the prey contents, then growth and division of the predator ultimately leading to new prey emerging. Further, Host Independent (HI) mutants are capable of growth in rich nutrients in the lab, with divergent morphologies observed. Some transcriptional studies have proved invaluable as a tool for studying some of these processes, but a complete lifecycle study is lacking. Here, we present a high-resolution transcriptional profile throughout the predation cycle giving insights to all of these various stages. For B. bacteriovorus to fulfil its promise as a novel antimicrobial agent, more needs to be understood about predation outwith the paradigm laboratory conditions. In order to address this, predation carried out on a multidrug-resistant clinical isolate of Serratia marcescens was subjected to transcriptional analysis, including predation by a B. bacteriovorus mutated by deletion of the global regulator DgcC, a strain incapable of HI growth. Cluster analysis provides an unbiased means of ordering data by calculating distances between datapoints in n-dimensions, allowing grouping of gene expression from different experiments. Here, I develop a pipeline for the analysis of B. bacteriovorus RNA-Seq data with cluster analyses to bring further insights to both unpublished work from our laboratory and by re-analysing datasets of published work by other groups. These analyses discover that groups of genes are tightly sequentially regulated throughout the predatory cycle, giving insight to their functions. They show that predation upon Serratia is significantly different from that of predation on E. coli, with different transcriptional profiles throughout the predation cycles. The response of B. bacteriovorus to exposure to pooled human serum seems to be one of protection from the antimicrobial elements in serum rather than metabolism of potential nutrients in the medium. The response to nutrient broth and non-prey Gram-positive Staphylococcus aureus surprisingly includes genes which are specific to Gram-negative prey modification, suggesting that the obligate predator B. bacteriovorus co-regulates predation and nutrient utilisation pathways. Many groups of genes are identified by analyses of mutant transcription as important in various regulatory pathways. Along with insights into the predation process and condition responses, the gene clusters generated in this project by novel re-analyses of data identify many targets for future projects to better understand the predation process and how B. bacteriovorus reacts in more clinically relevant conditions; a prerequisite for the fulfilment of its promise as a potential novel antimicrobial therapy

Transcriptional analysis of Bdellovibrio bacteriovorus responses to different prey and environments

Bdellovibrio bacteriovorus is a small, highly motile Gram-negative bacterium that preys upon other Gram-negative bacteria. It does this by burrowing through the outer layers of the prey cell and establishing itself in the periplasm, before consuming the prey contents and using these for growth as a filament. When the prey contents are depleted, this filament septates to produce new B. bacteriovorus progeny which burst out of the prey shell and go on to attack further prey. Its prey includes pathogens of plants, animals and humans, including multidrug-resistant pathogens which are emerging as a major health threat. Thus it has potential as a novel therapeutic to overcome the lack of new antibiotics. This prospect is particularly attractive as genetic resistance to B. bacteriovorus predation has not been demonstrated, rather a plastic resistance to a sub-population is seen, which when recovered remains as susceptible to predation as the parent population. There are many stages to this complex predatory lifecycle: swimming or gliding to search out potential prey, attachment to and detecting suitable prey, formation of, and entry into, a pore in the outer layers which is then re-sealed. Then killing and rounding of the prey is followed by staged degradation of the prey contents, then growth and division of the predator ultimately leading to new prey emerging. Further, Host Independent (HI) mutants are capable of growth in rich nutrients in the lab, with divergent morphologies observed. Some transcriptional studies have proved invaluable as a tool for studying some of these processes, but a complete lifecycle study is lacking. Here, we present a high-resolution transcriptional profile throughout the predation cycle giving insights to all of these various stages. For B. bacteriovorus to fulfil its promise as a novel antimicrobial agent, more needs to be understood about predation outwith the paradigm laboratory conditions. In order to address this, predation carried out on a multidrug-resistant clinical isolate of Serratia marcescens was subjected to transcriptional analysis, including predation by a B. bacteriovorus mutated by deletion of the global regulator DgcC, a strain incapable of HI growth. Cluster analysis provides an unbiased means of ordering data by calculating distances between datapoints in n-dimensions, allowing grouping of gene expression from different experiments. Here, I develop a pipeline for the analysis of B. bacteriovorus RNA-Seq data with cluster analyses to bring further insights to both unpublished work from our laboratory and by re-analysing datasets of published work by other groups. These analyses discover that groups of genes are tightly sequentially regulated throughout the predatory cycle, giving insight to their functions. They show that predation upon Serratia is significantly different from that of predation on E. coli, with different transcriptional profiles throughout the predation cycles. The response of B. bacteriovorus to exposure to pooled human serum seems to be one of protection from the antimicrobial elements in serum rather than metabolism of potential nutrients in the medium. The response to nutrient broth and non-prey Gram-positive Staphylococcus aureus surprisingly includes genes which are specific to Gram-negative prey modification, suggesting that the obligate predator B. bacteriovorus co-regulates predation and nutrient utilisation pathways. Many groups of genes are identified by analyses of mutant transcription as important in various regulatory pathways. Along with insights into the predation process and condition responses, the gene clusters generated in this project by novel re-analyses of data identify many targets for future projects to better understand the predation process and how B. bacteriovorus reacts in more clinically relevant conditions; a prerequisite for the fulfilment of its promise as a potential novel antimicrobial therapy

Chiasma

Newspaper reporting on events at the Boston University School of Medicine in the 1960s

Injections of predatory bacteria work alongside host immune cells to treat Shigella infection in zebrafish larvae

Bdellovibrio bacteriovorus are predatory bacteria that invade and kill a range of Gram-negative bacterial pathogens in natural environments and in vitro [ 1 and 2]. In this study, we investigated Bdellovibrio as an injected, antibacterial treatment in vivo, using zebrafish (Danio rerio) larvae infected with an antibiotic-resistant strain of the human pathogen Shigella flexneri. When injected alone, Bdellovibrio can persist for more than 24 hr in vivo yet exert no pathogenic effects on zebrafish larvae. Bdellovibrio injection of zebrafish containing a lethal dose of Shigella promotes pathogen killing, leading to increased zebrafish survival. Live-cell imaging of infected zebrafish reveals that Shigella undergo rounding induced by the invasive predation from Bdellovibrio in vivo. Furthermore, Shigella-dependent replication of Bdellovibrio was captured inside the zebrafish larvae, indicating active predation in vivo. Bdellovibrio can be engulfed and ultimately eliminated by host neutrophils and macrophages, yet have a sufficient dwell time to prey on pathogens. Experiments in immune-compromised zebrafish reveal that maximal therapeutic benefits of Bdellovibrio result from the synergy of both bacterial predation and host immunity, but that in vivo predation contributes significantly to the survival outcome. Our results demonstrate that successful antibacterial therapy can be achieved via the host immune system working together with bacterial predation by Bdellovibrio. Such cooperation may be important to consider in the fight against antibiotic-resistant infections in vivo

Characterizing the flagellar filament and the role of motility in bacterial prey-penetration by Bdellovibrio bacteriovorus

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

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

Prey killing without invasion by Bdellovibrio bacteriovorus defective for a MIDAS-family

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

Can Variations of (HNMR)-H-1 Chemical Shifts in Benzene Substituted with an Electron-Accepting (NO2)/Donating (NH2) Group be Explained in Terms of Resonance Effects of Substituents?

The classical textbook explanation of variations of (HNMR)-H-1 chemical shifts in benzenes bearing an electron-donating (NH2) or an electron-withdrawing (NO2) group in terms of substituent resonance effects was examined by analyzing molecular orbital contributions to the total shielding. It was found that the -electronic system showed a more pronounced shielding effect on all ring hydrogen atoms, relative to benzene, irrespective of substituent +R/-R effects. For the latter, this was in contrast to the traditional explanations of downfield shift of nitrobenzene proton resonances, which were found to be determined by the sigma-electronic system and oxygen in-plane lone pairs. In aniline, the +R effect of NH2 group can be used to fully explain the upfield position of meta-H signals and partly the upfield position of para-H signals, the latter also being influenced by the sigma-system. The position of the lowest frequency signal of ortho-Hs was fully determined by sigma-electrons.This is peer-reviewed version of the following article: Baranac-Stojanović, M. Can Variations of 1H NMR Chemical Shifts in Benzene Substituted with an Electron-Accepting (NO2)/Donating (NH2) Group Be Explained in Terms of Resonance Effects of Substituents? Chemistry - An Asian Journal 2018, 13 (7), 877–881. [https://doi.org/10.1002/asia.201800137]Supplementary material: [http://cherry.chem.bg.ac.rs/handle/123456789/3181